I) HORIZONTAL AGAROSE GEL ELECTROPHORESIS *
Gel Buffers: *
Preparation Of Agarose Gels *
Sample Preparation *
Running Gels *
Extraction of DNA from Low melting agarose Preparative Gels *
Quick Protocol for Standard Small Analytical Gel *
II) Hybridisation *
Southern Blotting; General Protocol *
DNA Hybridisation of Southern Blots; General Protocol *
Southern Blots; Biodyne B membrane *
DNA Hybridisation of Southern Blots; Biodyne B membrane *
Two Dimensional Electrophoresis Blotting Hybridisation-"Cross-Blotting" *
III) Sequencing *
Maxam & Gilbert Chemical Sequencing *
Old Maxam & Gilbert Sequencing Protocol *
Dideoxy-Sequencing *
Sequencing And S1-Mapping Gels *
I) HORIZONTAL AGAROSE GEL ELECTROPHORESIS
For double stranded DNA various percentages of agarose to running buffer (wt/vol) are used to obtain optimal molecular weight separation at different molecular weights.
Separation also depends to some extent on the running buffer used. The following should be used as a guide only.
|
Agarose % |
Molecular Weight Range |
Comments |
|
0.2% |
5-40 kb |
Gel very weak, high melting point (HM) agarose only should be used. Separation in 20-40 kb range greatly improved by increase in ionic strength of running buffer, eg Loening's 'E' buffer is much better than Tris-borate or Glycine buffers. |
|
0.4% |
5-30 kb |
With care both high (HM) and low (LM) melting point agarose gels may be used. Again separation around 20 kb improved at ionic strength. |
|
0.6% |
3-10 kb |
As for 0.4%. |
|
0.8% |
1-7 kb |
General purpose gel. Separation not greatly affected by choice of running buffer. Bpb runs at about 1 kb. |
|
1 % |
0.5-5 kb |
As for 0.8%. |
|
1.5% |
0.3-3 kb |
As for 0.8%. Bpb runs at about 500 bp. |
|
2 % |
0.2-1.5 kb |
HM agarose must be poured at about 80oC to avoid problems of rapid setting. |
|
3 % |
0.1-1 kb |
Separation of fragments as small as 50 bp can be acceptably accomplished. |
|
Over 2% gels |
HM agarose: 3% is probably the highest concentration which can be poured successfully. Gel plate should be prewarmed to 60oC and agarose poured at 90-95oC, i.e. directly from microwave. LM agarose: Pour at ~65oC to avoid problem of rapid setting. |
Gel Buffers:
1. Loening's 'E' Buffer
(36 mM Tris, 30 mM NaH2PO4, 1 mM Na2EDTA).
High ionic strength, not for preparative gels.
For 5 litres of 10x
218 g Tris
234 g NaH2PO4.2H2O
18.6 g Na2EDTA.2H2O
2. Glycine Buffer
Low ionic strength, very good for preparative gels but can also be used for analytical.
For 2 litres of 10x
300 g Glycine
300 ml 1 M NaOH (or 12 g pellets)
80 ml 0.5 M EDTA
3. Tris Borate Buffer
(90 mM Tris, 90 mM Boric Acid, 2.5 mM Na2EDTA). Low ionic strength, can be used for preparative and analytical gels.
For 5 litres of 10x
545 g Tris
278 g Boric Acid
46.5 g EDTA
4. Tris-Acetate Buffer
(40mM Tris-acetate, 0,4mM EDTA). Intermediate ionic strength, can be used for preparative and analytical gels but has poor buffer capacity.
For 1litre of 50x
242g Tris base
57.1 ml glacial acetic acid
37.2g Na2 EDTA.2H2O
HM agarose
As a general rule, dissolve agarose in water plus 1/10 volume of required 10x buffer by boiling on a hot plate with a magnetic stirrer in a volumetric flask covered with foil to prevent evaporation. Once it has been boiling for 2-3 min check the agarose has all dissolved and leave to cool to about 65oC preferably in a 65oC water bath. Add ethidium bromide to the required concentration (usually 0.5 ug/ml) from 10 mg/ml stock and pour onto a clean prepared gel plate on a level surface. Select pocket formers and leave gel to set for 15-30 mins.
The gel plate is glass with perspex side pieces. Masking tape or Scotch tape is used to seal the ends.
When gel is thoroughly set (can be noted by slight milky white appearance) the surface of the gel is covered with distilled water to facilitate re oval of the slot formers.
The sealing tape is then also removed. The gel is placed in the gel box and running buffer added to just cover the gel and pockets.
LM agarose
LM agarose is used for preparative gel electrophoresis usually in glycine buffer. After boiling as for HM agarose it is cooled to 37oC in a water bath and poured onto a gel plate at room temperature. The gel is then allowed to set at room temperature for upwards of an hour depending on thickness.
Gel and sample volumes
The size of gel and pocket used depend on sample volume and the amount of DNA to be fractionated. Usually the pocket volume should be twice the sample volume and the maximum amount of DNA loaded never greater than 10ug/cm2 of pocket face in the running direction.
Small gel plates are 11 cm x 12.5 cm and large gel plates 22 x 25 cm. For a 3 mm deep small gel, volume required is 11 x 12.5 x .3 = 41.25 ml, in practice make up about 50 ml). Pocket sizes for small analytical gels are 0.5 cm long, 0.1 cm wide and there is about a 0.1 cm gap between the bottom of the pocket and the gel plate. Thus the volume of a small analytical gel pocket is 0.5 x 0.1 cm x (0.3-0.1) cm = 0.01 or 10 ul. Sample volume should then not exceed 5 ul, see above. For sample volumes of 10 ul make up about 70 ml of gel.
Samples should be in a final volume l/2x that of the gel pocket, x less than mM NaCl or equivalent ionic strength and all divalent ions chelated with excess EDTA. Usually a 5x stock solution of loading buffer is made up and this added directly to the sample.
5x loading buffer for Analytical Gels
This is suitable for samples directly from most restriction digests.
50% v/v glycerol
100 mM Na-EDTA pH 8
0.125% w/v Bromophenol Blue (Bpb)
0.125% w/v Xylene Cyanol (XC)
(0.5% SDS may be included for better results)
5x loading buffer for Preparative Gels
The analytical loading buffer may be used but if the sample has been phenol extracted + precipitated, as would be done for best results, it may be dissolved in TE and the following simple loading buffer used.
50% v/v glycerol
0.l25% w/v Bpb
0.l25% w/v XC
Agarose analytical gels may generally be run at about 10 volts/cm. When fragments of 5 kb and above are to be analysed better results will however be obtained at 5 volts/cm or less. When fragments of 1 kb and less are analysed better results are obtained at higher volts/cm. For both large and small fragments it is often better to allow DNA to enter gel at lower volts/cm than will be used during the major part of the run.
Preparative gels, except when 300 bp or smaller fragments are to be prepared, should be run as slowly as is practival. It is often convenient to run O/N at between 1 and 2 volts/cm. At 2 volts/cm the Bpb dye will migrate at about 1 cm/hr regardless of gel percentage.
Extraction of DNA from Low melting agarose Preparative Gels
1. Run appropriate LM agarose gel in glycine or TBE buffer (but not E-Buffer).
2. Remove minimal gel slice with scalpel, whilst viewing gel under 360 nm UV source.
3. Dilute gel slice 1:1 with 1 M TNE.
4. Heat to 68oC in water bath for 10 mins, mix from time to time. Check carefully that gel is fully dissolved before continuing.
5. Allow to cool to ~37oC in water bath.
6. Extract with 1 volume of phenol-0.1% hydroxyquinoline (but see following note) saturated with 0.3 M TNE. Spin 10,000 rpm, 5 mins at R/T.
(Note: this is the orginal protocol and worked for many years with LM agarose from Sigma, BRL and ICN. Phenol/chloroform mixes did not work with these agaroses. In 1996, LM agarose purchased from Boehringer M. (BCL) did not work in the protocol with phenol extraction but did work when phenol/chloroform was used in its place.)
7. Remove S/N, carefully avoiding thick white interphase.
8. Repeat above extraction once or twice more, (this may not be necessary).
9. Extract once with 1 volume chloroform.
10. Precipitate DNA with 2-2.5 volumes EtOH. Leave more 30min to 1 hour at -20oC (best when very little DNA is present) or ~15-30 mins at -70oC.
11. Recover DNA by centrifugation and pour off S/N. Wash (without agitation) with 80% EtOH from freezer and immediately centrifuge to recover DNA. Dry and redissolve.
1. 63 ml H20, 7 ml 10x buffer + HM agarose.
2. Boil for 2-3 mins until dissolved.
3. Cool to 65oC.
4. Add 3.5 ul 10 mg/ml EtBr (NB. use gloves).
5. Pour and leave 30 mins.
6. Make samples to 10 ul in loading buffer.
Southern Blotting; General Protocol
(C.M. Read)
Stock Solutions
Denaturing solution:
|
0.5 M NaOH |
20g |
|
1.5 M NaCl |
87.66 g |
|
_______ |
|
|
Final volume |
1 litre |
Neutralising solution:
|
1 M Tris HCl pH 8.0 |
121.14 g |
|
1.5 M NaCl |
87.66 g |
|
_______ |
|
|
Final Volume |
1 litre |
Dissolve ingredients in H2O and adjust pH to 8.0 with HCl.
20x SSC:
|
3 M NaCl |
876.6 g |
|
0.3 M Na3Citrate |
441.15 g |
|
_______ |
|
|
5 litre |
Dissolve in H2O and adjust pH to 7.0 with NaOH.
1x SSC
Protocol
1. Run appropriate agarose gel with DNA samples to be blotted. Require:
0.5-1.0 ug or less of recombinant plasmid DNA digest
or 10 ug of mammalian DNA digest
2. After electrophoresis photograph the gel with a rule placed alongside marker DNA lanes.
3. Remove unused areas of gel (including marker DNAs) with scalpel.
4a. To aid the transfer of large DNA fragments incubate gel in a 5 fold volume excess of 0.25M HCl for 15 min. ONLY. Better is to break DNA by exposing to 1000 to 1500 mJ of light at 250nm in a Stratalinker or similar. This step can be omitted.
4b. Denature DNA by soaking the gel in 250 ml of Denaturing solution previously placed in a plastic tray. Gently rock tray at room temperature for 1 hour.
5. Carefully pour off Denaturing solution and replace with 250 ml of Neutralising solution. Gently rock at room temperature for 1 hour.
6. Meanwhile put a sponge in a plastic box and fill it with 1x SSC almost to the top of the sponge, making sure that all the air is excluded from the sponge. Place Whatman 3MM paper cut to the size of the sponge, on top of the sponge. Alternatively, support a glass plate on four feet (e.g. pierced 50ml tube caps) in an open tray (developing tray) and use two layers of 3MM as wicks placed across the plate. Exclude air bubbles between 3MM layers by smoothing out with a glass rod or pipette.
7. Invert the gel so that the underside is now uppermost, (facultative). Place the gel on the wet 3MM paper, making sure that there are no air bubbles trapped between.
8. To prevent liquid contact around gel, wrap Saran over the whole area. Using a scalpel cut round gel and remove the Saran covering the gel. Altenatively, frame gel with strips of Parafilm.
9. With gloves on and using Millipore forceps, remove the Genescreen, Hybond N or similar charged membrane or a Nitrocellulose membrane and cut with a scalpel to a size, in each direction, about 4 mm larger than the gel.
10. Float filter on the surface of a solution of 1x SSC until it wets completely from beneath. Then immerse the filter in the 1x SSC for a few minutes.
11. Place the wet filter square on top of the gel making sure that air bubbles are completely excluded.
12. Cut two pieces of Whatman 3MM paper to the same size as the gel. Wet these in 1x SSC and place them individually on top of the nitrocellulose filter, making sure that air bubbles are excluded once again.
13. Place a stack of paper towels (5-8 cm high) on top of the 3MM papers and then a glass plate to weight them down.
14. Allow transfer to proceed for 12-24 hours, less than 1 kb DNA fragments on a 0.8% gel transfer within 2 hours. 15 kb DNA takes greater than 15 hours.
15. Remove the towels and 3MM papers. Mark the positions of the gel slots (and left/right top/bottom orientation) on the filter with a biro.
16. Peel off the filter and soak it in 1x SSC for 5 mins. Drain off excess fluid and dry the filter on a sheet of 3MM paper at room temperature. The blotted gel may be removed from the sponge and stained in 0.5 ug/ml EtBr to visualise DNA fragments not transferred.
17. Place the filter between two sheets of 3MM paper and wrap this in foil. Bake for 2 hours at 80oC under vacuum. Alternatively, most charged membranes can be UV crosslinked using a 250nm germicidal lamp or a specialised crosslinker. Store filter at room temperature.
DNA Hybridisation of Southern Blots; General Protocol
(C.M. Read)
Stock Solutions
20 x SSC:
|
3 M NaCl |
876.6 g |
|
0.3 M Na3Citrate |
441.15 g |
|
__________ |
|
|
Final volume |
5 litres |
Dissolve in H2O and adjust pH to 7.0 with NaOH.
6x SSC: 300 ml 20x SSC per litre.
2x SSC: 100 ml 20x SSC per litre.
3x SSC, 0.1% SDS, 0.1% NaPyrophosphate:
150 ml 20x SSC, 5 ml 20% SDS, 1 g NaPyrophosphate per litre.
1x SSC, 0.1% SDS, 0.1% NaPyrophosphate:
50 ml 20x SSC, 5 ml 20% SDS, 1 g NaPyrophosphate per litre.
0.1x SSC, 0.1% SDS, 0.1% NaPyrophosphate:
5 ml 20x SSC, 5 ml 20% SDS, 1 g NaPyrophosphate per litre.
100x Denhardt’s Solution:
|
2% (w/v) Ficoll 400 (Pharmacia) |
20 g |
|
2% (w/v) Polyvinylpyrrolidone (Sigma) |
20 g |
|
2% (w/v) Bovine Serum Albumin (Sigma) |
20 g |
|
__________ |
|
|
Final volume |
1 litre |
Add Ficoll and Polyvinylpyrrolidone to H2O and dissolve by warming to 65oC. Cool on ice. Then add BSA and dissolve up. Adjust to final volume.
Prehybridisation Mix (for high copy no. probes):
|
6x SSC |
300 ml, 20x |
|
10x Denhardt’s |
100 ml, 100x |
|
0.1% SDS |
5 ml, 20% |
|
0.1% NaPyrophospha |
1 g |
|
____ |
|
|
1 litre |
Add 20x SSC and 100x Denhardts to ~900 ml H2O, then add SDS and Pyrophosphate. Adjust to final volume.
Prehybridisation Mix (for single copy no. probes):
|
6x SSC |
300 ml 20xSSC |
|
5x Denhardt’s |
50 ml 100xDenhardt’s |
|
0.1% SDS |
5 ml 20% |
|
0.1% NaPyrophosphate |
1 g |
|
10% Dextran Sulphate |
100 g |
|
100 ug/ml denatured Salmon sperm DNA |
10 ml, 10 mg/ml |
|
_____ |
|
|
1 litre |
Hybridisation Mix:
This consists of Prehyb. mix with EDTA added to 10 mM and denatured nick translated probe DNA (by heating in a 100oC water bath for 10 mins). Require about 50 ul of mix/cm2 of bag or 10 ml for short and 20ml for long hyb. bottles. Do not have more than ~2 x106 cpm/ml and for Hybond N of 1996 onwards even less, see booklet, as this increases the background dramatically.
Hybridisation
1. Float the baked filter on the surface of 6x SSC until it wets from beneath. Immerse the filter in the 6x SSC for a few minutes.
2. Insert the filter into a plastic-bag or better a hybridisation bottles. (See Note).
3. Add ~0.2 ml of Prehyb mix (prewarmed to 65oC) for each square centimetre of bag or 10ml for small 20-25ml large hybridisation bottles. Squeeze as much air as possible from the bag and then seal it. Incubate the bag at 65oC in a box in a shaking water bath or hybridisation bottles in rotary oven for at least 4 hours.
4. Open the bag by cutting off one corner with scissors. Squeeze out as much prehyb mix as possible.
5. Using a pasteur pipette add the Hyb mix (prewarmed to 65oC). Squeeze out the air and seal the bag up again.
6. Incubate the bag at 65oC in a box filled with water in a shaking water bath for at least 24 hours.
7. Remove Hyb mix from bag with a pasteur pipette. Remove filter using forceps and transfer to a plastic tray containing 250 ml of 3x SSC, 0.1% SDS, 0.1% NaPyrophosphate. If using bottles leave filter in bottle and wash there. Wash for a few minutes at room temperature and repeat.
8. Filters are then washed at 65oC for 1 hour in a shaking water bath to successively lower SSC concentrations to give the required level of stringency.
250 ml of 3x SSC, 0.1% SDS, 0.1% Napyrophosphate (low level)
250 ml of 1x SSC, 0.1% SDS, 0.1% Napyrophosphate (medium)
250 ml of 0.1x SSC, 0.1% SDS, 0.1% Napyrophosphate (high)
9. The filter is finally washed with 250 ml of 2x SSC for a few minutes at room temperature and then dried at room temperature on a sheet of Whatman 3MM paper.
10. Autoradiograph filter using Fuji RX film with intensifying screen at -70oC.
Note
Best results are usually obtained with hybridisation bottles. Hybridisation may, however also be carried out in plastic sandwich boxes, but in this case the volume of Prehyb and Hyb mixes should be increased to ~50 ml.
Re-use of filter:
Either; submerge membrane in water, 0.1%SDS, bring to boil and allow to cool to R/T for 30-60 min. (nitrocellulose) or; 30 min at 45oC in 0.4M NaOH. Transfer to 0.1 x SSC, 0.1% SDS, 0.2M tris-HCl, pH 7.5 at 45oC for a further 30 min.
Southern Blots; Biodyne B membrane
Stock Solutions
0.4 N NaOH
Protocol
1. Run appropriate agarose gel with DNA samples to be blotted. Require:
0.5-1.0 ug or less of recombinant plasmid DNA digest
or 10 ug of mammalian DNA digest
2. After electrophoresis photograph the gel with a rule placed alongside marker DNA lanes.
3. Remove unused areas of gel (including marker DNAs) with scalpel.
4. For best transfer break DNA by exposing to 1000 to 1500 mJ of light at 250nm in a Stratalinker or similar.
5. Immediately prepare the transfer as for General Southern Blot protocol, steps 6 to 16, using 0.4 N NaOH as tranfer medium. No pretreatment of the gel is necessary. Fixation by baking or UV is unnecessary. Stored filters damp or after blotting with 3MM, at 4oC in Saran.
DNA Hybridisation of Southern Blots; Biodyne B membrane
Solutions:
Hybridisation solution;
|
0.5 M NaxHyPO4, pH 7.2 |
25 ml |
|
20% SDS |
17.5 ml |
|
0.5M Na-EDTA, pH neutral |
50 ul |
|
______ |
|
|
Final volume |
50 ml |
Prehybridisation solution; Hybridisation solution plus 150 ug/ml s.s. DNA
Wash I;
|
0.5 M NaxHyPO4, pH 7.2 |
40 ml |
|
20% SDS |
250 ml |
|
______ |
|
|
Final volume |
1 l |
Wash II;
|
0.5 M NaxHyPO4, pH 7.2 |
40 ml |
|
20% SDS |
50 ml |
|
______ |
|
|
Final volume |
1 l |
Protocol:
Steps are best performed in a rotating hybridisation oven. 65oC is used throughout.
1. Place membrane at in prehybridisation solution for 15 min. or more.
2. Remove hybridisation solution and replace with hybridisation solution. Previously, boil probe DNA for 5 min. and add as a small volume to hybridisation solution.
3. Hybridise overnight.
4. Prewarm the Wash solutions to 65oC.
5. Rinse membrane rapidly in Wash I to remove excess probe.
6. Wash 3 to 4 times in Wash I, 15 min, each time.
7. Wash once or twice in Wash II. It may be necessary to increase temperature to 70 or 75oC or dilute Wash II by 3 or 10 fold to obtain higher stringency washes.
8. The filter is finally washed with 250 ml of 2x SSC for a few minutes at room temperature and then blotted on a sheet of Whatman 3MM paper.
9. Autoradiograph the filter enclosed in Saran using Fuji RX or similar film with intensifying screen at -70oC.
Two Dimensional Electrophoresis Blotting Hybridisation-"Cross-Blotting"
(U.A. Patel)
Used for restriction mapping and identification of internally repetitive sequences in plasmids etc.
Stock Solutions
Stock solutions are as for normal Nick Translation and Southern Blotting.
Protocol
1. Add in the following order:
100 uCi [a32P] - dCTP (3200 Ci/mMole, NEN)
5 ul Nick Translation Buffer
4 ul Cold dNTP's
X ul 500 ng restricted DNA
Y ul H2O
0.5 ul E. coli Polymerase I
___________
30 ul Final volume
2. Incubate mixes at 14oC for 90 mins.
3. Terminate reaction by adding:
10 ul 3 M NaCl
10 ul 500 mM EDTA
50 ul dH2O
to give final volume of 100 ul and concentrations of 300 mM NaCl, 50 mM EDTA.
4. Load each terminated reaction mix onto a 2 ml SPC 50 column, equilibrated in 0.3 M TNE. Elute with 0.3 M TNE, collecting 8 fractions of which the first is 250 ul in volume and the rest 100 ul in volume.
5. Remove 1 ul aliquots from each fraction and Cerenkov count (3H channel in LS counter). Nick translated DNA will normally appear in fractions 5 and/or 6. Specific activities ranged between 1.5-2.2 x 108 dpm/ug DNA depending how nicked the cosmid DNA was.
First Dimension-Southern Blotting Of Unlabelled DNA
1. Make an appropriate agarose gel (Loenings). 275 ml volume poured onto 22x25 cm size gel plate with 10 cm wide slot for sample. (Up to 250 ul of sample volume can be loaded).
2. Either load: 5 ug of restricted cosmid DNA
or 50 ug of restricted genomic DNA
3. Electrophorese at 20V O/N.
4. Electrophorese until RNA run ~9.5 cm. Then photograph gel with a rule placed alongside marker DNA lanes.
5. Remove unused areas of gel (including marker DNAs) with scalpel (gel size 10x 9.5 cm).
6. Denature DNA by soaking gel in 250 ml of denaturing solution (0.5 M NaOH, 1.5 M NaCl) previously placed in a plastic tray. Gently rock tray at R/T for 1 hour.
7. Carefully pour off denaturing solution and replace with 250 ml of neutralising solution (1 M Tris HCl pH 8.0, 1.5 M NaCl). Gently rock at R/T for 1 hour.
Meanwhile put a sponge in a plastic box and fill it with 10x SSC almost to the top of the sponge, making sure that all the air is excluded from the sponge. Place Whatman 3MM paper cut to the size of the sponge, on top of the sponge. Exclude air bubbles by smoothing out with a glass rod.
8. Invert the gel so that the underside is now uppermost. Place the gel on the damp 3MM paper, making sure that there are no air bubbles trapped between.
9. Wrap Saran over the whole area. Using scalpel cut round gel and remove the saran covering gel.
10. With gloves on and using millipore forceps, remove the nitrocellulose (Genescreen) filter and cut with a scalpel to a size, in each direction, about 4 mm larger than the gel (eg 10.4x9.9 cm).
11. Float filter on the surface of 2x SSC solution until it wets completely from beneath, then immerse filter in for a few mins.
12. Place the wet filter square on top of the gel making sure that air bubbles are completely excluded.
13. Cut two pieces of Whatman 3MM paper to the same size as the gel (10x9.5 cm). Wet these in 2x SSC and place them individually on top of the nitrocellulose filter, making sure that air bubbles are excluded again.
14. Place a stack of paper towels (5-8 cm high) on top of the 3MM papers and then a glass plate to weight them down.
15. Allow transfer to proceed for 16-24 hours.
16. Remove the towels and 3MM papers. Mark the positions of the gel slots (and left/right, top/bottom orientation) on the filter with biro.
17. Peel off the filter and soak it in 6x SSC for 5 min. Drain off excess fluid and dry the filter on a sheet of 3MM paper at room temperature. (Avoid any light bulbs to dry filters).
18. Place the filter between two sheets of 3MM paper and wrap this in foil. Bake for 2 hours at 80oC under vacuum. Store filter at room temperature.
Prehybridisation Of Unlabelled DNA Blots
1. Float the baked filters on the surface of 6x SSC until it wets from beneath. Immerse the filter in the 6x SSC for a few mins.
2. Filter placed individually in box containing 50 ml prehybridisation mix, prewarmed to 65oC.
3. Prehybridise at 65oC in a shaking water bath for at least 4 hours.
Prehybridisation mix:
6x SSC
0.1% SDS
0.1% Na Pyrophosphate
10x Denhardts
100 ug/ml denatured Salmon sperm DNA
Second Dimension-Electrophoresis Of Labelled DNA
1. Make an appropriate agarose gel (Loenings). 275 ml volume poured onto 22x25 cm size gel plate with 10 cm wide slot for sample (up to 250 ul of sample volume can be loaded).
2. Load nick translated restricted DNA (using gloves, screen and eye goggles).
3. Electrophorese at 20V overnight in the hot room.
4. Electrophorese until marker DNA has run the same distance as the first dimension gel because filter and gel size will have to be identical.
5. Unused areas of gel (including marker DNAs) removed with scalpel behind screen (gel size 10x9.5 cm).
6. Denature DNA by soaking gel in 250 ml of denaturing solution (1.5 M NaCl, 0.5 M NaOH) previously placed in a tray. Gently rock tray at room temperature for 1 hour.
7. Carefully pour off denaturing solution (in hot room sink) and replace with 250 ml neutralising solution (1 M Tris HCl pH 8.0, 1.5 M NaCl). Gently rock at room temperature for 1 hour.
Cross Blot Hybridisation
1. While the second dimension gel is neutralising, put a sponge in a plastic box and fill it with prehybridisation mix (without the salmon sperm DNA), prewarmed to 65oC, making sure that all the air is excluded from the sponge. Place a Whatman 3MM paper cut to the size of the sponge, on top of the sponge. Exclude air bubbles by smoothing out with a glass rod.
2. Invert the hot gel so that the underside is now uppermost. Place the gel on top of the damp 3MM paper, making sure that there are no air bubbles trapped between. Also make sure that the orientation of the gel is known.
3. Wrap saran over the whole area. Using scalpel cut round gel and remove the saran covering gel.
4. Place the prehybridised filter of the first dimension (DNA side down) on top of the second dimension gel with the directions of electrophoresis perpendicular to each other.
NOTE: Here you can put up to 4 first dimension filters, one on top of each other, on top of the second dimension gel.
5. Cut two pieces of Whatman 3MM paper to the same size as the gel (10x9.5 cm). Wet these in 2x SSC and place them individually on top of the nitrocellulose filter, making sure that air bubbles are excluded again.
6. Place a stack of paper towels (5-8 cm high) on top of the 3MM papers and then a glass plate to weight them down.
7. To reduce evaporation seal the outside of the plastic box by taping the saran to the box.
8. Carry out cross-blot hybridisation in an oven at 65oC for between 16-24 hours.
9. Remove the towels and 3MM papers. Mark the positions of the gel slots (and left/right, top/bottom orientations) on the filter(s) with biro.
10. Peel off filter(s) using forceps and transfer to plastic tray containing 500 ml 3x SSC, 0.1% SDS, 0.1% Na pyrophosphate. Wash for a few mins at room temperature.
11. Filter(s) then washed individually stepwise for 1 hour in a shaking waterbath at 65oC in the following solutions to obtain the required stringency.
(a) 250 ml 3xSSC, 0.1% SDS, 0.1% Na pyrophosphate (low level)
(b) 250 ml 1xSSC, 0.1% SDS, 0.1% Na pyrophosphate (medium)
(c) 250 ml 0.1xSSC, 0.1% SDS, 0.01% Na pyrophosphate (high level)
12. The filter(s) are finally washed in 500 ml of 2x SSC for a few minutes at room temperature and then dried at room temperature on a sheet of 3MM paper.
13. Autoradiograph filter(s) using Fugi RX film with intensifying screen at 70oC.
Maxam & Gilbert Chemical Sequencing
from Lab of A. R-C, 12/8/94
Use 1ug carrier DNA/reaction
Some reagents are extremely toxic, therefore use gloves and a hood where appropriate.
N.B. i) DNA must be clean and salt free. It should be EtOH precipitated and resuspended in water.
ii) DMS should be disposed of in 5M NaOH
iii) Hydrazine should be disposed of in 3M FeCl3
|
G+A |
|
DNA in 30 ul H2O Add 1 ul piperidine formate (1M, pH2) Incubate 25 min (short DNA; 45 min oligos; 15 min long DNA) at 37 oC Add 150 ul fresh 1 M piperidine (1:10 from bottle) (plus 1 ug tRNA) Keep at 0oC |
|
C+T |
|
DNA in 10 ul H2O Add 15 ul hydrazine (mix, do not vortex) Incubate R/T 10 min (short DNA: 30 min oligos; 5 min long DNA) (add 1ug tRNA, 1ul) Add 1 ml n-butanol, mix very well, spin 1 min Add 150 ul 1 M piperidine to pellet, Keep at 0 oC |
|
C |
|
DNA in 7 ul H2O Add 20 ul 5 M NaCl, mix Add 30 ul hydrazine, Mix gently (do not vortex) 5 min R/T (10 min shorter DNA, 30 min oligos) 200 ul hydrazine stop, 1ug tRNA 750 ul EtOH, cool -80 oC, Spin 10 min 225 ul H2O, 25 ul 3 M NaAc 750 ul EtOH, cool -80 oC, Spin 10 min Wash 80% EtOH 100 ul H2O 1 ml n-butanol, mix , spin 1 min Add 150 ul 1M piperidine to pellet Keep at 0 oC |
|
G |
|
100 ul 50 mM sodium cacodylate buffer (pH8) 100 ul fresh DMS solution (1 ml cacodylate buffer + 10 ul DMS) 1 min 20 oC (2 min oligos; 0.5 min long DNA) 50 ul stop buffer, 1ug tRNA (750 ul ETOH, cool -80 oC, spin 1 min) (100 ul H2O) 1 ml n-butanol, mix, spin 1 min 100 ul H2O 1 ml n-butanol, mix, spin 1 min Add 150 ul 1M piperidine to pelletKeep at 0 oC |
|
To all samples90 oC for 30 min (Use water bath on specially protected rack)Cool to R/T1.2 ml n-butanol, mix, spin 1 min150 ul 1% SDS, vortex, change tubes!!!1 ml n-butanol, mix, spinadd 3 ul H2O, wait 5 minadd 3 ul formamide bufferwarm at 90 oC with caps opened for 5 minCool in ice and load onto gel. |
Solutions:- (If tRNA carrier used, add same day)1M piperidine formate4% v/v formic acid in H2O adjusted to pH2.0 with piperidine1 M piperidine (1:10 dilution in water from bottle)Cacodylate buffer50mM Sodium cacodylate1 mM EDTApH 8.0DMS stop1.5 M NaAc, pH 7.01.0 M -mercapto-ETOH(100 ug/ml tRNA)Hydrazine stop0.3 M NaAc, pH 7.00.1 M EDTA(25 ug/ml tRNA)1% SDS in H2OGOOD LUCK
Old Maxam & Gilbert Sequencing Protocol
(R.F. de Winter)
G reaction:
1. Pipette in 1.5 ml eppendorf:
200 ul 50 mM Na-cacodylate, pH 8.0, 1 mM EDTA
5 ul DNA (50-100x 103cpm)
1 ug carrier DNA
2. Mix and chill on ice. Add 1 ul DMS, mix, and incubate for 4 min at
+20oC.
3. Stop reaction with:
50 ul 1.5 M NaAc, pH 7.5, 1.0 M mercaptoethanol
5 ug tRNA (carrier)
750 ul cold abs. EtOH
4. Precipitate 5 min at -70oC.
5. Spin 5 min. in eppendorf centrifuge. Remove supernatant and place it in
5 M NaOH.
6. Redissolve in:
250 ul 0.3 M NaAc (0oC)
750 ul cold abs. EtOH
7. Precipitate 5 min at -70oC. Wash with 80% EtOH and vacuum dry.
8. Redissolve in freshly diluted 1 M Piperidine, (stock: 10 M), and take
up in capillary which is then sealed on both ends.
G+A reaction:
1. Pipette in 1.5 ml eppendorf:
10 ul distilled H2O
5 ul DNA (50-100x103cpm)
1 ug carrier DNA
2. Mix and add 2 ul 1.0 M Piperidine formate, pH 2.0. Mix and incubate for
80 min. at 20oC.
3. Add 5 ug tRNA and lyophilize. Redissolve in 20 ul dH2O and again
lyophilize.
4. Redissolve in 20 ul freshly diluted 1 M Piperidine (stock: 10 M), and
take up in capillary which is sealed on both ends.
C+T reaction:
1. Pipette in 1.5 ml eppendorf:
10 ul distilled H2O
5 ul DNA (50-100x103cpm)
1 ug carrier DNA
2. Mix and add 30 ul Hydrazine. Mix and incubate for 12 min. at 20oC.
3. Then add:
200 ul 0.3 M NaAc, 0.1 mM EDTA
5 ug tRNA
750 ul cold abs. EtOH
4. Precipitate at -70oC for 5 min.
5. Spin 5 min. in eppendorf centrifuge. Pipette supernatant into 2 M FeCl2.
6. Redissolve in:
250 ul 0.3 M NaAc
750 ul cold abs. EtOH
7. Precipitate at -70oC for 5 min.
8. Spin 5 min. in eppendorf centrifuge. Wash with 80% EtOH and vacuum dry.
9. Redissolve in 20 ul freshly diluted 1 M Piperidine (stock: 10 M), and take
up in capillary which is sealed on both ends.
C Reaction:
1. Pipette in 1.5 ml eppendorf:
15 ul 5 M NaCl
5 ul DNA (50-100x103cpm)
1 ug carrier DNA
2. Mix and add 30 ul Hydrazine. Mix and incubate for 10 min. at 20oC.
3. Then add:
200 ul 0.1 mM EDTA
5 ug tRNA (carrier)
750 ul cold abs. EtOH
4. Precipitate at -70oC for 5 min.
5. Spin 5 min. in eppendorf centrifuge. Pipette supernatant into 2 M FeCl2.
6. Redissolve in:
250 ul 0.3 M NaAc
750 ul cold abs. EtOH
7. Precipitate at -70oC for 5 min.
8. Spin 5 min. in eppendorf centrifuge. Wash with 80% EtOH and vacuum dry.
9. Redissolve in 20 ul freshly diluted 1 M Piperidine (stock: 10 M), and
take up in capillary which is sealed on both ends.
From Here All Four Reactions Receive The Same Treatment
1. Incubate capillaries at 90oC for 30 min. Transfer contents into 1.5 ml
eppendorf tube containing 20 ul destilled H2O.
2. Lyophilize (appr. 60 min.). Redissolve in 20 ul destilled H2O.
3. Lyophilize (appr. 30 min.). Redissolve in 20 ul destilled H2O.
4. Lyophilize (appr. 30 min.). Determine number of cpm of each sample.
Redissolve and equalise samples finally in:
80%(v/v) formamide
10 mM NaOH
1 mM EDTA
0.1%(w/v) xylene cyanol
0.1%(w/v) bromophenol blue
Times for reactions can be varied. Last time I have been using: G Reaction: 3 min.; A Reaction: 80 min.; C+T Reaction: 10 min.; C Reaction: 8 min.
Reference:
Maxam, Allan M., and Gilbert, Walter. (1980). Sequencing End-Labelled DNA with Base-Specific Chemical Cleavages. In: Methods in Enzymology 65, 499-559.
Isolation Of Mini-Prep Plasmid DNA For Sequencing
1. Decant 2,5 ml O/N bacterial culture into a eppendorf tube, spin for 2 minutes. Completely remove the supernatant by aspiration.
2. Resuspend the pellet in 200 ul GTE (50mM glucose, 10mM EDTA, 25mM Tris-HCl pH8.0) solution and vortex.
3. Add 400 ul 0.2 N NaOH/l% SDS and invert several time before placing on ice for 5 minutes. Do not vortex!
4. Add 300 ul 3 M K acetate (pH 4,8), invert several times and place on ice for 7 minutes. Do not vortex!
5. Centrifuge for 5 minutes and decant (simply pour) the supernatant into another tube. (Here a phenol/chloroform followed by a chloroform extraction can be included, but is not necessary for sequencing reaction). Add 700 ul absolute ethanol, mix (vortex very well) and spin immediately for 5 minutes.
6. Discard the supernatant and wash the pellet with l ml 70% ethanol. Spin 5 minutes, drain tube well and vacuum dry the pellet.
7. Resuspend the DNA pellet in 30 ul TE buffer. For restriction enzyme digestion take 2-4 ul (depending how big the insert is).
Sequencing
Take 5-8 ul of the plasmid prep., add 8-11 ul water and 4 ul l N NaOH/0,1 mM EDTA, mix and incubate for 5 minutes at 80-99 oC, we usually use 99oC. Add 60 ul ETOH and 2,2 ul 2M ammonium acetate, pH4.6. Vortex and store at -20 oC >30 minutes or O/N, (or l5 minutes in dry ice/ethanol bath). Spin l0 minutes and vacuum-dry the pellet. Resuspend in l0 ul water, add 2 ul annealing buffer and 2 ul primer. Proceed according the instructions of the sequencing kit.
Alternative Rapid Denaturation & Annealing:
5ul DNA, (EXACTLY) 1ul 1N NaOH/0.1mMEDTA and 1pmol primer, 10ul total volume. 80-99 oC for 5min, (earlier protocols used 22 or 65 oC). Add (EXACTLY) 4 ul TDMN and leave at R/T for 10 minutes. Use directly in sequencing reaction as annealed template.
TDMN:
0.28M TES (free acid)
0.12M HCl
0.05M DTT
0.08MMgCl2
0.2M NaCl
Note: prepare this and NaOH solution with maximum precision, i.e. using graduated flasks.
Ref: Jones, D.S. and Schofield, J.P. NAR vol. 18, No. 24, 7463-64 (with some modifications)
Sequencing And S1-Mapping Gels
(D.R. Smith and C.M. Read)
40% Acrylamide solution:
|
Acrylamide |
38 g |
|
Bis-acrylamide |
2 g |
|
_________ |
|
|
final volume |
100 ml |
Solution is made up and then filtered through a paper or preferably a 0.45um filter. Store at 4oC.
10x TBE buffer:
|
Tris base |
108 g |
|
Boric acid |
55 g |
|
Na2EDTA.2H2O |
9.3 g |
|
_____ |
|
|
final volume |
1 litre |
This gives a stock solution of pH 8.3
1. Use two 25 cm plates for S1-mapping gels
50 cm plates for sequencing.
2. Clean plates thoroughly:
1x with detergent
1x with distilled water
Lay plates on bench and wipe dry.
Wash plates 1x with 70% Ethanol
1x with absolute Ethanol
Wipe dry each time.
One plate only may be siliconised by wiping or spreading on 1% dichlorosilane in CCl4. Mark siliconised plate for future identification!
3. Take two 0.25 mm spacers, lay on one plate and sandwich them between the two plates. Tape together the bottom and sides of the plates. Put the side clamps on. Lay the plates almost horizontal.
4. For 8% polyacrylamide TBE urea gels, add the following together:
|
For 25 cm long gels |
For 50 cm long gels |
|
|
Urea |
21 g |
42 g Urea |
|
10x TBE |
5 ml |
|
|
40% acrylamide soln. |
10 ml |
20 ml |
|
_____ |
_____ |
|
|
final volume |
50 ml with H2O |
100 ml with H2O |
Dissolve at 37oC and cool to room temperature. Then 0.45um filter and add:
|
375 ul 10% APS |
750 ul 10% APS |
|
|
20 ul TEMED |
40 ul TEMED |
Mix gently by swirling and pour smoothly into the plate sandwich. (As an aid to pouring place a tape across the top of the notched plate to give a wider pocket).
5. Put in the Teflon comb, and lay the gel sandwich flat. Gel should set
in about 30-60 min.
6. When gen is set, remove bottom tape and set up in electrophoresis apparatus, with 1x TBE in reservoirs.
7. Rinse gel pockets immediately using a syringe.
8. Pre-electrophorese at either ~30 W (25 cm gels) or ~55 W (50 cm gels).
9. Samples in Formamide dye mix are boiled for 2 min, prior to immediate loading into freshly rinsed-out gel pockets.
10. Electrophorese at either ~30 W (25 cm gels) or ~55 W (50 cm gels).
11. After the run has terminated switch off the power supply, remove the gel from the power source and lay flat on a bench. Remove the side pieces and carefully separate the two plates with the aid of a fine spatula. Ensure that the gel is stuck to only one gel plate.
Autoradiography depends upon the type of radioactivity that has been used for label.
After electrophoresis the gel is prepared for autoradiography. It is necessary to prevent diffusion of the DNA bands either by "fixing" i.e. by precipitating the DNA with acetic acid, or by performing the autoradiography at -70oC. There are several autoradiographic methods available.
Low Temperature Autoradiography (for phosphorus-32)
This is the simplest method of autoradiography for sequencing gels labelled with phosphorous-32. The gel is transferred from the glass plate to a piece of 3MM paper. This is done by laying a sheet of 3MM onto the gel, carefully smoothing the paper down and then peeling the paper off. The gel should be transferred onto the paper. The gel then covered with Saran wrap and smoothed down. As the gel is 50 cm long and the cassette only 40 cm long, it is necessary to remove the top 10 cm of the gel. This is best carried out with either a very sharp, new scalpel blade, or a clean sharp pair of scissors. The gel can now be transferred to the cassette and a piece of film overlaid. If required an intensifying screen can be used although this will reduce resolution of the final autoradiograph. The closed cassette is then placed in a -70oC freezer.
Autoradiography Of Fixed and Undried Gels
Fixing the gel in acetic acid often makes autoradiography at low temperature unnecessary.
Open the gel plates as above and carefully lower the gel plate with the gel on top into a tank containing a solution of 10% methanol, 10% acetic acid. Allow to fix for a minimum of 10 minutes. Drain the gel and when sufficiently drained, carefully lay a piece of 3MM onto the surface of the gel. Smooth down and carefully peel off the 3MM paper. The gel should come with the 3MM paper. Lay the paper flat on the bench and cover with a piece of Saran wrap. Trim the gel to the correct size. Autoradiograph at room temperature.
Autoradiography of Dried Gels
(for high resolution with 32P and essential with 35S)
Drying the gel before autoradiography gives improved definition and is essential for . The gel must be dried if the label used is 35S as the beta-particle does not have sufficient energy to escape even a thin layer of wet gel.
When using a gel drier, the early treatment is the same as above, i.e. fixing the gel and then transferring onto a piece of 3MM paper. The trimmed gel is placed on the gel drier and the gel is then dried for up to an hour depending on the thickness of the gel. After drying do not cover the gel with Saran wrap. Autoradiograph overnight at room temperature.
Quick & Dirty for Sequencing gels
0.25 to 0.5 mm thick gels are dried directly with no pretreatment to fix or remove urea. Can give marks on film.