DNA manipulation and labeling *

LABELLING OF DNA FRAGMENTS (including filling out) *

Dephosphorylation of DNA ends *

5' End Labeling (5'-overhang) *

5' End Labeling (3'-overhang, blunt end or "difficult" end) *

3' End Labeling (Using Calf Thymus Terminal Transferase) *

3' End Labeling (Using T4 DNA Polymerase) *

Filling-out/resecting unpaired DNA ends; *

Biotinylation of DNA and fixation on S.A. agarose *

Biotinylation: *

Binding to S.A particles: *

Nick translation *

Random Priming *

Removal of excess label *


DNA manipulation and labeling



LABELLING OF DNA FRAGMENTS (including filling out)

(R.F. de Winter)

Dephosphorylation of DNA ends

This can be done either;

Directly after digestion with restriction enzyme by adding 1/100 volume 1M Tris-HCl ph 9.0 and 1 to 5 units of Calf Intestinal Alkaline Phosphatase. Incubate for 1 hr. at 37oC.

Or; For better results, Phenol/Chloroform and chloroform extract the DNA and precipitate after restriction. Redissolve in TE pH 8.3. Add 1 to 5 units of Calf Intestine Alkaline Phosphatase (CIAP). Incubate for between 1 and 14 hr at 37oC, longer times are better. phenol/chloroform then Chloroform extract and precipitate the DNA before restricting with a second enzyme.

Dephosphorylation of DNA ends that are going to be labelled using the denaturation method (see below) should be done with Bacterial Alkaline Phosphatase in a similar way to CIAP but at 65oC for 1 hr.

5' End Labeling (5'-overhang)

10x PNK buffer:

500 mM Tris HCl pH 7.6

100 mM MgCl2

50 mM DTT

100 mM Spermidine (pH 7.0)

1. Aliquot and mix together:

1-2 ul

10x Polynucleotide Kinase (PNK) buffer

1 pmole

dephosphorylated DNA ends


[g 32P]-ATP

1 unit

T4 Polynucleotide Kinase

x ul




10-20 ul

Final volume

2. Incubate at 37oC for 30 min.

5' End Labeling (3'-overhang, blunt end or "difficult" end)

1. Prepare dephosphorylated DNA solution (at 1 pmole/ul) in:

10 mM Tris HCl pH 9.5

1 mM Spermidine

0.1 mM EDTA

2. Denature the DNA for 3 min. at 100oC, quickly chill on ice and then rapidly add 1/10 volume of:

500 mM Tris-HCl pH 9.5

100 mM MgCl2

50 mM DTT

several units Polynucleotide Kinase

g32P -ATP to final 1 uM minimum

3. Incubate at 37oC for 30 min.


1. Van de Sande, J.H., Kleppe, K. and Khorana, H.G. Biochem., 12, 5050-5055 (1973).

2. Lillehang, J.R., and Kleppe, K. Biochem., 14, 1225-1229 (1973).

3' End Labeling (Using Calf Thymus Terminal Transferase)

1. Aliquot and mix:

1 pmole

DNA end

1 ul

5 mM CoCl2

1 ul

1M Na-cacodylate

1 ul

BSA (1 mg/ml)

50 uCi

[a 32P]-rNTP

2-5 units

Terminal Transferase (Sigma)



10 ul

Final volume

2. Incubate at 37oC for 1 hr.

3. Denature Terminal Transferase by incubation for 10 min. at 68oC, then add to the sample:

1 ul

0.5 M EDTA

80-90 ul

1x TE

500-1000 units

RNAase A (for [a 32P]-CTP and [a 32P]-UTP),

RNAase T1 (for [a 32P]-GTP)

or RNAase T2 (for [a 32P]-ATP or GTP)


100 ul

Final volume

4. Incubate at 37oC for 1 hr.

3' End Labeling (Using T4 DNA Polymerase)

1. Incubate 1 pmole of phosphorylated DNA ends with 1-2 units of T4 DNA polymerase, with no nucleotides present for 10 min. at 37oC in (essentially ligase buffer which can be used less the ATP instead):

50 mM Tris HCl pH 7.5

10 mM MgCl2

10 mM DTT

2. Add three cold dNTPs at 30 to 100mM each (e.g. 1/10 vol of a 0.3 to 1 mM stock) and 25-30 uCi of the required [a 32P]-NTP. Incubate for 30 min. at 37oC.

3. Chase the reaction by adding 1/10 volume 1 mM solution of all four dNTPs and incubate for another 30 min.

Filling-out/resecting unpaired DNA ends;

Using T4 polymerase to fill-out/resect unpaired DNA ends is essentially the same as 3’ labeling but the four nucleotides are added immediately at 100mM each. 3' ends will be resected, 5' ends will be filled out.


Biotinylation of DNA and fixation on S.A. agarose

Digest the plasmid with the 3 prime end restriction enzyme (and fill the end with Kleenow if necessary).

Digest with a restiction enzyme producing a 5 prime overhang containing a T residue. Purify the band of interest on LMP agarose.


x ul DNA fragment (20 pmol)

15 ul 10X react 2 (BRL)

7,5 ul Biotin-7-dATP 0,4 mM (BRL cat.:19509-017)

7,5 ul dGTP, dCTP, dTTP 1mM

5 ul Klenow fragment (20 U) (BRL)

1 ul 32P dCTP (as a tracer)


150 ul total, 3 hrs at 37 oC

Pass the reaction two times on Sephadex G-50 packed in a pasteur pipette (to get rid of free nucleotide).

Estimate the efficiency of labelling by Cerenkov counting (1 uCi (2.2 x 106dpm) is about 1 to 1.7x106 Cerenkov cpm).

Binding to S.A particles:

Wash the resin 3 times with TNE 100.

Incubate overnight at 4 oC with biotinylated DNA fragment adjusted at 100 mM NaCl.

Wash 3 times with TNE 1000.

Calculate binding by comparing resin counts before and after the final wash, or by comparing the counts of the DNA added and the count of the wash (magnetic S.A. particles quench a little bit the radiocativity).


Nick translation

(C.M. Read)

Stock solutions

100 uM cold dNTPs:

10 mM dATP

1 ul

10 mM dTTP

1 ul

5 mM dGTP

2 ul



Final volume

100 ul

Nick Translation Buffer:

300 mM Tris HCl pH 7.5

300 ul 1 M

30 mM MgCl

30 ul 1 M



Final volume

1 ml total volume

E. coli Polymerase I:

7.5 u/ul Polymerase I (Biolabs).

DNase I:

0.1 ng/ul DNase I (Boehringer) in 1.5 mg/ml BSA.


1. Add in the following order:

20 uCi

[a-32P] -dCTP

1 ul

Nick Translation Buffer

0.8 ul

Cold dNTPs

x ul

100 ng DNA

y ul


0.1 ul

Polymerase I

0.4 ul




6.0 ul

Final volume

(Reaction mixture can be scaled up.)

2. Incubate mixes at 14oC for 90 mins.

3. Terminate reaction by adding:

10 ul

3 M NaCl

10 ul

500 mM EDTA

74 ul


to give final concentrations of 300 mM NaCl and 50 mM EDTA.

4. Load each terminated reaction mix onto a 1.8 ml SPC50 column, equilibrated in 0.3 M TNE. Elute with 0.3 M TNE and collect 10 fractions of 100 ul volume, see Removal of Excess Label.

5. Remove 1 ul aliquots from each fraction and Cerenkov count (3H channel in liquid scintillation counter). Nick translated DNA will elute in fractions 5 to 7.

A test reaction using 100 ng of pCRI DNA gave ~20 x 106 cpm in total (specific activity of 3 x 108 dpm/ug DNA.

Random Priming

Removal of excess label

All unincorporated 32P- nucleotide label is separated from the labelled DNA molecules by passing the reaction mixture, diluted to 100 ul with TE, through a 1.8ml pasteur pipette column of SPC-50 (Pharmacia) in 0.3 M TNE or G50 (Pharmacia, Mol Biol grade) in TE. Collect 10x 100 ul fractions. Count fractions directly in scintillation counter. Pool and precipitate the DNA peak, fractions 5 to 7, with 2-2.5 volumes of cold EtOH.

Note: SPC50 is a derivatised G50 which strongly binds most nucleic acid-binding proteins and but shows exceptionally low non-specific binding of nucleic acids. It must be used at an ionic strength of around 0.3M or higher.



(Ian. T. Kennedy, pers. comm. 11/'79)

I. Enzyme Mix:

2 mg/ml glycerophosphate dehydrogenase (1 ml)

100 ul

2 mg/ml triosephosphate isomerase (1 ml)

1 ul

10 mg/ml glyceraldehyde-3-P dehydrogenase (2 ml)

20 ul

10 mg/ml 3-phosphoglycerate kinase (1 ml)

2 ul

5 mg/ml lactate dehydrogenase (2 ml)

20 ul

All enzymes are used as ammonium sulphate precipitates as delivered from Boehringer-Mannheim.

This mix can be stored at 4oC for months.

II. Prepare solutions (which must be made fresh each time):

a) Dissolve 4.4 mg Na-pyruvate in 1 ml H2O (40 mM)

b) Dissolve 47 mg. cysteine-HCl in 5 ml H2O. Use this solution to dissolve 70 mg Trizma, pH should be approx. 8.

Remember, the above solutions should be fresh!

III. Prepare mix A:

500 mM Tris-HCL pH 9

50 ul

100 mM DTT

30 ul

2.4 mM L-a-glycerol phosphate

25 ul

20 mM b-NAD

25 ul

300 mM MgCl2

20 ul

2 mM Na-ADP

12.5 ul

40 mM Na-pyruvate (see II)

12.5 ul

From stocks

10x L-a-glycerol phosphate:

40 mg/ml (mw: 406.4) (?? Check)

10x b-NAD:

143 mg/ml (mw: 708.5)

10x Na-ADP:

10 mg/ml (mw: 478.0)

IV. Enzyme solution: immediately before use.

1. Resuspend enzyme mix by inverting tube several times.

2. Centrifuge 15 ul enzyme mix in Eppendorf tube, 3 min. in microcentrifuge.

3. Using a drawn out capillary remove the supernatant as completely as possible.

4. Dissolve in 15 ul 50 mM Tris-HCl pH 9.

V. Prepare Mix B: immediately before use

Cysteine solution

50 ul


10 ul

0.5 M Tris pH 9

7 ul

Enzyme solution

5 ul

VI. Reaction:

1. To 15 mCi 32P-orthophosphate in 150 ul H2O at room temperature (remove from 4oC storage more than 1 hr before reaction) add 80 ul Mix A

2. Remove 0.5 ul to use as marker for chromatography and add this to 10 ul ATP carrier solution (10mM unlabeled ATP, 20 mM KH2PO4)

3. Add 20 ul Mix B. Mix well by rotating tube and incubate at room temperature.

VII. After 10 min:

1. Remove 0.5 ul and add to 10 ul ATP carrier solution

2. Spot onto PEI thin layer plate (of about 8cm long by 2 cm wide only) beside an equal amount of the marker, (approx. 0.5 ul each).

3. Develop chromatogram in saturated tank in 0.75 M KH2PO4

4. Expose plate to film for 1 - 2 min.

5. Allow reaction incubation to continue for a total of 30 to 40 min.

VIIIa. If conversion is greater than 90%:

1. Add H2O to 0.4 ml

2. Transfer to conical graduated centrifuge tube

3. Rinse reaction tube with 2x 200 ul aliquots of H2O and transfer to centrifuge tube

4. Total volume should be about 0.8 ml.

VIIIb. If conversion after 10 min. is incomplete:

1. Make fresh enzyme mix, fresh enzyme solution and add 1ul of this to reaction. Check as in VII. if still incomplete:

2. Make fresh A and B mixes and add to reaction:

150 ul H2O

80 ul A

20 ul B

3. Continue as in VII.

IX. Extraction and Storage:

1. Add 0.8 ml phenol:chloroform (1:1)

2. Cover tube with Saran

3. Vortex thoroughly, then spin

4. Remove top phase with Eppendorf pipette

5. Repeat extraction 3 times with peroxide-free ether (1 ml)

6. Add 1 volume abs. EtOH, mix thoroughly and store at -20oC

X. Analyse ATP:

1. Mix 5 ul of ATP preparation with 5 ul ATP-P1 carrier solution.

2. Spot 0.5 ul on PEI, run chromatogram and autoradiograph.

3. Dilute 5 ul of the above mixture in 500 ul H2O and count 10 ul to determine the stock mCi/ml