ml
Sterile Water
5 ml 10X Nick Translation Buffer
5 ml EACH 10X dNTP Stocks (d
CTP, dATP, dTTP; 10X = 0.3mM concentration)
ml DNA (concentrated to at least 0.125 ug/ul), normally 0.5- 1.0
mg
20 ml Alpha 32P dGTP label (aqueous; at 10mCi/ml) 200 mCi/mg DNA
1 ml DNA Polymerase I (We use New England Biola
bs at 15U/ml)
1 ml DNase, at 1:1000 dil'n (amount varies with the size of DNA, amount is used for
a 1200 bp fragment)
50 ml FINAL VOLUME
2) Proceed to Step 2 below.
METHOD II (Kit):
1) Add the following in order to a sterile, silanized, 0.5 ml microfuge tube:
-
ml Sterile Water (use to QS to 50 ml)
&
nbsp; 5 ml |
10X Nick Translation Buffer with dNTPs
ml DNA (concentrated to at least 0.125 ug/ul), normally 0.5 -1.0 mg
20 ml |
Alpha 32P-dGTP labeled nucleotide (10mCi/ml); 200 mCi/mg DNA.
1 &nbs
p; ml DNA Polymerase I/DNAse mix
50 ml FINAL VOLUME
2) Incubate the mixture for 1.5 hours at 15°C in a heat block placed in a cold room to control temperature.
3) Prepare a G-50 (Fine) Sephadex colum
n in a 5 ml, sterile, plastic serological pipet in the following manner:
- Cut 5 – 6 mm off the bottom of the pipet by scoring a circle around the tip with a scalpel, then breaking off the end with a hemostat. This will enable you to fit an 18 gauge needle on the end of the pipet.
- Pack an 18 gauge sterile needle with some silanized, autoclaved glass wool and connect this to the bottom of the pipet. Make sure the wool is in the needle hub and clip off any excess.
Add about 1 foot of tubing to the end of the needle. Also snap off the top mouthpiece of the pipet by hand.
- Clamp the tubing with a hemostat, add some sterile water to the column, unclamp, and allow to flow through to remove air bubbles, this is best achieved by thumping the pipet but be sure to hold the needle or it may fly off. Leave about 1 ml of water in the column when finished. Pipet a 50% gel slurry of G-50 (equilibrated in 50mM EDTA, 10mM Tris-HCl, pH 7.5 with 0.1% v/v sodium az
ide added to prevent bacterial contamination) into the column with a sterile, glass, Pasteur pipet. Be sure not to introduce air bubbles. Release hemostat and allow the column to pack. When finished settling, the gel bed should be no more than 1 cm from the top of the pipet. This will ensure easy loading of the sample with a Pipetman and allow room for buffer space at the top of the pipet. Approximately 7 ml of buffer will flow through.
- Wash the column with a couple of bed volumes of n
ick translation elution buffer. This will equilibrate the column with the SDS and get rid of the sodium azide, which is believed to be inhibitory to various chemical reactions.
4) At the end of the incubation period, add the DNA sample to the gel bed with a sterile yellow Pipetman tip (be sure to drain all buffer on top of the gel bed first, as you do NOT want to dilute the sample). Allow the sample to penetrate the gel (this only takes a few seconds) and then chase two times with a sm
all amount of buffer. After this chase, you may fill the column up with buffer. Don't allow the column to run dry!
5) Monitor the progression of the labeled DNA through the column with a hand-held Geiger counter (it will come off in the FIRST peak). By the time the sample reaches the middle of the column, you should be able to detect two distinct peaks. The second peak contains unincorporated 32 P along with smaller DNA fragments (broken down material) and will be much hotter (~2X) than the
first peak. If you cannot detect two peaks, then the nick translation has bombed and you must start over again!
6) Start collecting the labeled DNA when it is half way through the tubing as determined by the Geiger counter. Collect in a sterile, snap-cap, polypropylene, Falcon culture tube (12 X 75mm) or a 1.5 ml microfuge tube. The peak should come off in a relatively small volume, approximately 0.75-1 ml. Collect until the counts drop off, keeping the volume reasonably small however.
7) After you have eluted your sample, remove 1 ul of it and smear on the bottom of a scintillation vial. Add 5 mls of scintillation cocktail. Count for 1 minute in a scintillation counter to determine the activity, then calculate the specific radioactivity based on how much DNA you started out with and the total volume it is in:
[(CPM/ml) x total volume] / (mg DNA)
A successful labeling will give you at least 108 CPM/ ug of DNA. We usually obtain close to 2 X 108 CPM/mg.
RECIPES
10X Nick Translation Buffer (Meth
od I: 0.5M Tris-HCl, pH 7.5; 0.1M MgSO4; 10mM dithiothreitol; 500 mg/ml BSA)
For 1 ml, add the following:
500 ml of 1M Tris stock
100 ml of 1M MgSO4
10 ml of 1M DTT
10 ml of BSA (BRL 50mg/ml sterile stock)
380 ml autoclaved water
Keep frozen at -30°C. When the specific activity starts to drop, make up fresh buffer first.
Nick Translation Elution Buffer: (10mM Tris-HCl, pH 7.5; 50mM EDTA; 0.2% SDS)
For 100 ml:
10 ml 0.5M EDTA
1 ml 1M Tris-HC
l stock
2 ml 10% SDS
87 ml autoclaved water
Store at room temperature.
G-50-80 Sephadex (Sigma Chemical Company #G-50-80).
Swell the Sephadex in water for several hours, then equilibrate with several aliquots (equal volumes)
of 50mM EDTA, 10mM Tris-HCl, pH 7.5. Add sodium azide to 0.1% (v/v). Store at room temp.
2'Deoxyadenosine 5'Triphosphate, Disodium salt (Sigma #D6500)
2'Deoxycytidine 5'Triphosphate, Sodium sal
t (Sigma #D4760)
Thymidine 5'Triphosphate, Sodium salt (Sigma #D8635)
Make a 20mM stock solution for each dNTP by adding sterile water directly to the contents of each vial containing the crystalline powder. Then, make a 1/66.7 dilution for each to bring the final concentration to 0.3mM, which = a 10X working stock. This is much easier than weighing out minute quantities on the balance (risking inaccuracies); the 20mM stocks can be kept frozen (-30
6;C) for future use. The 10X (0.3mM) stocks should also be kept frozen at -30°C and thawed out slowly on ice when needed.
Tubing: Tygon micropore S-54-HL (ID 0.04 in.; O.D. 0.07 in.; wall 0.15 in.).
Original draft prepared by Renee Besta (1984) and revised by Mark Barton Frank (1987).
Copyright 1993, 1996, 1997, and 1998 by Mark Bar
ton Frank, Ph.D.
Proper citation for data acquired from this document is: "Besta, R. M. and Frank, M. B. Nick Translation Protocol. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/htl.html). 1997. Oklahoma City. Revision Date: September 18, 1998."
The following tables may be useful in preparing components of the nick translation reaction and keeping track of radioactiv
e waste.
NICK TRANSLATION REACTION METHOD 1
Individual components
Date:___________________ By: ___________________ |
Probe: _____________
____________________________ Date Prepared:__________
Add the following in order to a sterile, silanized, 0.5 ml microfuge tube:
ml
Sterile Water
5 ml 10X Nick Translation
Buffer
5 ml EACH 10X dNTP Stocks (dCTP, dATP, dTTP; 10X = 0.3mM concentration)
___ ml DNA; Concentration ____________ Amount _______(mg)
___ ml 32P d__TP; Referen
ce Date ___________; __________ Ci/mmol
___ ml Polymerase I
___ ml DNAse
50 ml FINAL VOLUME
Incubation Time: from ________ to ________ Temp: ________
Specific Activity:
_________
_ cpm ________ ml
Volume of sample collected ___________ml
Total cpm ____________ Cpm /mg DNA _________
Record of Use:
# Decay Vol
Date Days Factor CPM Out Volume Out Remaining Disposal Initials
NICK TRANSLATION REACTION
Method II (BRL Kit)
Date
:___________________ By: ___________________ |
Probe: _________________________________________ Date Prepared:__________
___