Sequencing problems!! - homopolymeric region and slippage (Jan/14/2005 )
I have some problems with sequencing a homopolymeric (poly A/T) region. With both primers the effect of the polymerase slippage during sequencing reaction ) is visible and leads to not readable sequences. I have tried a two step program (55 °C) and reduced the amplicon amount, but without a any results! I can not create a new primer because then I will not detect my wanted section any more. Cloning would be a solution, but not for me.
So does anybody have a good advice for me? What can I try next?
AB: BigDye Termination Kit 1.1
amplicon size 210 bp
Please try to reduce the extension temp to 60-65C.
Hello! I have already tried an extension at 50 °C and even 55 °C (two step: 10s 96°C/ 2 min 50 °C). Ich have made a hot start pcr to get a better amplicon, The gel ist good, but the sequencing reaction ist still bad. AB says 12 A/Ts are not much, but the kit-polymerase does not get over it.
Does the chromatogram become unreadable not long after your repeat tract?
Are you able to try ABI's Big Dye Terminators v3.0? I know that is good for repeat tracts. I have tackled poly A tracts with no problem.
You could try increasing the extension time from 2 minutes to 6 minutes with the two step protocol. Also increasing the number of cycles from 25 to 30 or 35 helps as well.
This should allow proper read through of your tract, otherwise I have been told to try and use DMSO to resolve the possible secondary structure, but i didn't need to resort to that!
Good luck! May the sequencing gods look upon you favourably!
The sequencen becomes unreadable directly after the tract (with both primers).
That means 80 % is unreadable with the rev-primer and 20 % with the for-primer. Unfortunatly I need the sequence 5 bp after the tract, performed with the rev-primer. The for-sequence just covers the wanted sequence and gets unreadable after this.
I can not try BDT 3.0/3.1 because it is not suitable for the ABI 310.
How much DMSO did you did you put in?
I perform a 10 µL-reaction in a standard (PE 9600) thermocycler, so how much do I have to us?
THX a lot!
Can you recommend a good book for troubleshooting and understanding the subject more, just for an advanced freshman?
I remember it was 5% DMSO. I was told to do this by technical help at ABI.
here is a site I just found with regard to a protocol involving DMSO.
Just use anchored primer will do. For example: 21Ts end with a degenerated base-V (5'tttttttttttttttttttttv3') on your polyA template. Use 3x primer concentration in your cycle sequencing rxn = 3.2pmole x 3 = 10pmole of anchored primer.
Before this, you must make sure there is only 1 polyA or poly T region within your template.
You have a real problem. From what you have described it appears that the problem is coming from the PCR step not the sequencing step (basically the PCR is generating a mix of templates). You have three possible solutions:
1. Gel purify your PCR product on a denaturing PAGE gel so that you can isolate only the one sized fragment. This works well provided your PCR product is not too large.
2. Clone and sequence multiple plasmids.
3. Try using a polymerase with reduced slippage. NEB sells a high processivity polymerase (fusion something) that might help.
Longer DNA sequencing reads
I don't understand your comment about BDT 3.1 not being suitable for the ABI 310 sequencer. We have been using it with that machine for many years. You may need to load a new mobility file, but ABI has those on its web site.
I agree that an anchored primer, as suggested, will work. I have done exactly that in the same situation, and it has functioned well. You won't read sequence 5 bp downstream from the slipped region, however.
I believe my sequence was slipping as a result of the PCR used to create the template fragment. You might want to try cloning the fragment (also suggested above, I believe) or using a more robust PCR enzyme (Finnzyme/NEB Phusion comes to mind).
You may be encountering problems due to two regions being amplified which are slightly different. Have you thought of that possibility? Or, alternatively, two regions both of which are primed by your sequencing primer.