Short and failed sequencing reads - (Jul/19/2007 )
I ahve been trying (and failing!) to sequence a 5kbp insert for a few weeks now. All of my sequencing comes back either failed or with very short reads. I tried the primers on genomic DNA and it worked fine so I don't think they are the problem. I was thinking maybe it is a problem with my plasmid DNA.
I am using TOP10 cells, pBS plasmid, and have been using the QIAGEN miniprep kit to purify. When run on a gel to check concentration I don't have much DNA, is this a fault with my purification or with my cells?
It has also been suggested there may be a secondary structure that is stopping the sequencing so I could cut my DNA with a RE first, does this seem plausible?
Also, this may be a silly question, but I am regrowing from glycerol stock (streaking onto plates, picking a single colony and growing overnight) is this an ok method and can I keep doing this? This won't inroduce any mutations or anything?
Thanks for the help, I'm a sequencing virgin!
Are you sure that you have enough DNA in the sequencing reaction?
I'm not entirely sure how to check, I just estimate from the gel how bright the band is compared to the markers and usually put the maximum amount into the reaction (5ul) . If I don't have enough DNA in my samples how do I make it more concentrated?
check DNA amount by OD measure.
Then if your DNA is too low concentrated you can precipitate it by either NaAcetate/ethanol or isopropanol or butanol, followed by wash in etOH70%.
Then resuspend in lower quantity of water.
I've succesfully sequenced shRNA constructions so i wouldn't get on secondary structure hypothesis first.
it's quite curious that a quiagen miniprep didn't gave you enough plasmid...
The culture volume can be doubled for increasing amount of total plasmid.
Are you sure of your homogenization ? critical step for cell lysis...
I once had the same problems with sequencing a plasmid. And it was because my DNA was not concentrated enough. I solved the problem by not sequencing the plasmid but a PCR product of my insert using my plasmid as template along with the sequencing primers. Never had any problems again. Maybe you can try the same.
Thanks for all the suggestions, I'm going to try them all out and hopefully one will work!