DNA manipulation and labeling *

(R.F. de Winter)

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.

10x PNK buffer:

1. Aliquot and mix together:

1-2 ul	10x Polynucleotide Kinase (PNK) buffer
1 pmole	dephosphorylated DNA ends
25-30mCi	[g 32P]-ATP
1 unit	T4 Polynucleotide Kinase
x ul	H2O
--------
10-20 ul	Final volume

2. Incubate at 37oC for 30 min.

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

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

3. Incubate at 37oC for 30 min.

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.

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):

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.

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).

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).

(C.M. Read)

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	H2O
0.1 ul	Polymerase I
0.4 ul	DNAse I
-------
6.0 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
74 ul	H2O

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.

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)

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

This mix can be stored at 4oC for months.

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

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.

Cysteine solution	50 ul
H2O	10 ul
0.5 M Tris pH 9	7 ul
Enzyme solution	5 ul

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