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5' end labeling by t4 pnk?
for some reason i'm getting extremely variable labeling efficiency (1000-fold) labeling oligos from 18-mer to 70-mer, which should not form significant secondary structures. i'm using neb t4 pnk, neb t4 pnk buffer, and gamma p32 atp from perkin-elmer. after 1 hour at 37 degrees, i buffer exchange / remove unincorporated nucleotides on an amersham g25 column. does anyone else experience this? is nebuffer 4 better for labeling? or could the columns be defective? also, since the columns are around $3 a piece after tax and shipping, does anyone know of a cheaper source or alternative?
thanks for the suggestion. i purified the oligos on a 16 cm 12% denaturing page before labeling and later ran them on a 10% denaturing page. they are full-length.
Have you tried running your hot oligos out on a ~10% polyacrylamide + urea gel to see what % of the products are full length? I know when I was labelling oligos for nuclease protection assays I had variability as some oligos were poorly synthesized and had a low % of full length product. If you run them out, you should only need a few minutes of exposure on film to see the laddering pattern and determine which oligos may not be any good.
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NEW HERAEUS 0141 1006 MERCURY LAMP 821934 PERKIN ELMER $75.00 Time Remaining: 2h 5m Buy It Now for only: $75.00 |
Perkin Elmer HS40XL Gas Chromatograph Headspace Sampler HS40 XL HS 40XL $982.42 Time Remaining: 2h 40m |
Perkin Elmer Cetus DNA Thermal Cycler 480 $315.00 Time Remaining: 3h 52m Buy It Now for only: $315.00 |
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Perkin Elmer 560 Atomic Absorption Spectrophotometer + $1,999.99 Time Remaining: 4h 13m Buy It Now for only: $1,999.99 |
Perkin Elmer Harvester Evac Head Kit With Instructons Drawing $599.99 Time Remaining: 4h 23m Buy It Now for only: $599.99 |
PERKIN ELMER PACKARD COBRA II AUTO GAMMA COUNTER E5005 $2,500.00 Time Remaining: 4h 50m Buy It Now for only: $2,500.00 |
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Perkin Elmer Thermogravimetric Analyzer TGA7 $8,250.00 Time Remaining: 4h 59m Buy It Now for only: $8,250.00 |
Perkin Elmer LS 55 Luminescence Spectrometer $4,975.00 Time Remaining: 5h 4m Buy It Now for only: $4,975.00 |
Perkin Elmer Series 200 LC Micro Pump Liquid Chromatography UNTESTED $24.99 Time Remaining: 5h 4m |
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