i am running a dna sequencing reaction in a 40 cm long gel but i am not able to get crisp bands.bands seems diffuse. running condition 1500 V / 1x TBE buffer. oligo length 24 base 5 end FAM albelled .I am attaching a picture below.what would be the possible cause.
General recommendations that can help:
1. Check for the absence of secondary structures, such as hairpins…
2. Purify your stock (labeled) oligo on 15-20% small urea-PAGE before doing sequencing & use.
3. Perform 32P labeling quickly (15’incubation) and do not keep labeled DNA stock unfrozen for a long time.
4. No salt (NaCl, KCl, MgCl2…) in loading buffer – only 10 mM EDTA (10 mM Tris) & blue dyes. Perform phenol(/chloroform) extraction followed by chloroform extraction and glycogen precipitation before loading on the sequencing gel.
5. Pre-run the sequencing gel and run it hot (~60°C).
24 base oligo is 5 ` end fam labelled. I purify the band using native page
-Native, (instead of urea) PAGE, can be also fine but better use urea PAGE.
- what about the other four points?
if the 15% you mention is the acrylamide concentration that is too high. $-8% is fine for sequencing. If using a shark tooth comb make sure the gel is well set ( it can take up to 2 hours if the APS is getting old...use fresh APS is best)and the comb firmly held between the plates while the gel is setting. wash the top of the gel well with buffer and when you reverse the comb do not push the teeth in at all far...just touching the surface of the gel is good. Pre run to heat the gel and rinse the wells thouroughly with running buffer then load the samples. Run at about 45-70 watts as running too fast causes distortion. of bands
Dear Varun you can try with 20 % denaturating Urea PAGE .
It seems you are running at very high voltage you can try at 80- 100 V.
I performed 20% denaturating UREA PAGE for separation of 22 mer using FAM labelled oligo and I got good result.
Hi Varun
1) Might me high salt conc. could be reason.
2) Clean The wells after solidification. Pre run the gel and again clen the wells properly with the running buffer.
i per run the gel for 1 hours before loading the sample. ALKALINE loading buffer contains 80 percent formamide and 10mM NaOH. just a additional infos, I am doing this reaction for dms footprinting. oligo stock is 5 months old (stored at -20 in a dark appendorf)but i make the reaction fresh and immediately load.Does that make a difference.i will wash the wells next time before loading the samples to see any change.i usually run at 1500V (23 watts 18mA)
You can follow Prof.Dimitar suggestion but You can perform with FAM labelld oligos also.
Instead of alkalineloading buffer you can try 2X loading buffer(90% formamide, 0.5% EDTA, 0.1% xylene cyanol and 0.1% bromphenol blue)
Before loading the samples pre run for 30 min-60 min.
Deanture the samples before loading usnig 2X loading buffer at 80-90° C for 10 min.
Wash the wells before pre run and loading samples to remove urea.
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