PCR AMPLIFICATION OF LONG DNA FRAGMENTS *
RT-PCR with a specific sequence primer *
First Strand Synthesis *
PCR reaction *
_________________________________________________________
PCR protocols
PCR AMPLIFICATION OF LONG DNA FRAGMENTS
10x PCR 3+ buffer:
(Maria R. Ponce and Jose L. Micol. NAR (1992), Vol. 20, No. 3, 623)
|
Tricine pH 8.4 |
300 mM |
|
MgCl2 |
20 mM |
|
Mercaptoethanol |
30 mm |
|
Gelatin |
0.1% |
|
Thesit |
1% |
Reaction:
|
10x PCR buffer |
10 ul |
|
dNTPs 2 mM each |
10 ul |
|
DNA (1ng/ul) |
5 ul |
|
Forward primer (10 pmoles/ul) |
5 ul |
|
Reverse primer (10 pmoles/ul) |
5 ul |
|
Taq DNA polymerase (5u/ul) |
0.5 ul |
|
_____ |
|
|
H2O to final reaction vol. |
100 ul |
RT-PCR with a specific sequence primer
Naz Islam 1996
Buffers:
5X First stand buffer [250 mM TRIS-HCl pH 8.3, 375 mM KCl and 15 mM MgCl2]
0.1 M DTT
10 mM dNTP mix (10 mM each dATP, dCTP, dGTP and dTTP)
SUPERSCRIPT RT (BRL) (200 U/µL)
10x PCR 3+ Buffer
Care must be taken to use only double (or single 45 min.) autoclaved solutions and pipettes.
1) poly A+ RNA (1 to 2µg) or total RNA (5 µg max.) x µl
Primer (random primer 75ng, oligo-dT 500ng or degenerate primer 200ng) x µl
H2O x µl
____
Final reaction volume 20 µl
2) Heat the mixture to 70oC for 10 min. and quick chill on ice. Collect the contents of the tubes by brief centrifugation and add the following:
|
5X First strand buffer |
4.0 µl |
|
0.1M DTT |
2.0 µl |
|
10 mM d(NTP) mix |
1.0 µl |
|
[a32P]-dCTP (3000 Ci/mMol) |
1µCi |
|
______ |
|
|
Final volume |
18.0 ml |
Mix the contents of the tube, briefly centrifuge and equilibriate the temperature by placing the vial at 37oC for 2 min.
3) Add 2.0 ml (200 U/ml) SUPERSCRIPT RT, mix gently and incubate at 37oC for 1h.
4) Place the tube on ice to terminate the reaction.
5) Remove 2 ml of the reaction and transfer to a microcentrifuge tube containing: 48 ml of 20 mM EDTA and 5 ml of yeast RNA (1 mg/ml)
6) To the remaining 18 ml add 6 ml 5M NaCl and 76 ml H2O. Extract once with 100 ml of phenol-chloroform 1:1 and once with chloroform alone.
7) Precipitate nucleic acid by adding 2.5 vol. EtOH, leave at -20oC for >1hr, spin 17 krpm, 20 min. (Sorvall SS34). Carefully remove S/N with pipette (keep in separate tube), add 200 ml 80% EtOH, -20oC, spin 10min 17 krpm and again carefully remove all S/N.
8) Dry pellet for 2 min. in SpeedVac and redissolve in 10 ml of H2O.
Analysis of cDNA products (first strand)
Incorporation:
Dot 10 ml of the above mixture on DEAE-filter paper and transfer to a glass vial. For control, dot equivalent activity of [a-32P] dCTP and transfer to a separate glass vial. Repeatedly, wash the filter paper by adding fresh 0.5 M NaH2PO4 until no significant radioactivity remains on the control filter. Determine radioactivity and calculate yield by the following equation:
Amount of = (CPM) X(50 ml/ 10 ml) X (20 ml/2 ml) X (4 pmol dNTP/ pmol dCTP)
cDNA (mg) S.A (CPM / pmol dCTP) X (3,030 pmol dNTP / mg cDNA)
Specific activity (S.A) = CPM / 10 ml
200pmol dCTP / 10 ml
Gel analysis:
Precipitate DNA from remaining 40 ml of synthesis reaction withdrawn for analysis by adding 20 ml of NH4OAc and 2.5 vol. of EtOH. Wash the ppt. 1X with 70 % EtOH, dry and dissolve in 9 ml of TE. Add 1 ml of 1N NaOH. Apply sample on 1.4 % agarose gel after adding 2.0 ml of 5X loading buffer use end labelled l HIND III markers. Dry gel post electrophoresis and autoradiograph.
|
10x PCR 3+ buffer |
10.0 ml |
|
2 mM d(NTP) mix |
10.0 ml |
|
cDNA |
10.0 ml |
|
forward primer (50pM/ml) |
2.0 ml (about 1 mg of a 25 mer) |
|
reverse primer (50pM/ml) |
2.0 ml (about 1 mg of a 25 mer) |
|
______ |
|
|
Add H2O to Final volume |
100.0 ml |
Finally add 0.5 ml Taq DNA polymerase (5U/ml), mix carefully and spin, add 100ml mineral oil and begin PCR reaction:- 1min 94.5o C, then 35 cycles 30sec 94.5o C, 30sec 37o C, 30sec 72o C and finally 10 min at 72 o C.