Really strange problem with sequencing and plasmid purification!
Posted 11 January 2011 - 07:56 AM
I´ve been having problems with sequencing for already 2 months and its really exchausting. The problem began as I said 2 motnhs ago when I cloned 3 different inserts (coding for different proteins) into the target vector (pGEX). I verified the correct colonies with colony PCR and after plasmid purification I ran another PCR just to be sure I didnt get any false positives. I ran 6 sequencing reaction (3 different plasmids with different inserts, reverse and forward rections for each), from which 4 were quite successful. The sequence of the first plasmid was perfect, I got the right clone. The second one was read only until 400 bp from both sides (the insert is 1500 bp) but atleast then I ran a BLAST analysis it cofirmed that the insert is the correct one. And after that all the problems began worsen. Just for information, when I prepare dna for sequencing I dont do phenol/chloroform extraction because my superviser doesnt want me to. So what I do is I usually do a RNAse treatment and after that purify dna through the gel extraction columns. And this has until now worked really well for me. So after that I thought that maybe these plasmids were not pure enough, so I transformed these to DH5alfa and purified again. Sent to sequencing to another facility and now I got sequences with multiple peaks. Then I decided to do the cloning again, which I also repeated several times. I have already like whole box of positive clones (verified all with PCR after purification) and all the sequencing results have multiple peaks and no readable sequences. Then I started to suspect that maybe my RNAse is out of date, I centrifuged it and found that something was growing inside of it. After that I got the new RNAse and just tried again with my first clones, which were allomost suffessful and did a RNAse treatment with this new RNAse and purified again, sent to sequencing and again multiple peaks (and nanodrop show the 260/280 ration 1,84 and 230/260 around 2,2 - so it seems to be quite nice)! So now I dont know anymore. The primers are working well, the clones are the correct ones, the sequencing method itself is ok (I have done sequencing reaction myself and I have also sent to other lab which does all the preparations), I have had two allmost successful sequencing and even after replacing the RNAse I still dont get any good results. I dont understand could it be somekind of contamination (DNAse for example)? And how could it influence my plasmids which once have already been sequenced with quite promising results. Any suggestions or ideas would be really helpful. I will replace also all my solutions and maybe I will even try to do purification with different pipettes. But I would really like to know what kind of strange thing is that?
Thank you very much in advance!
Posted 11 January 2011 - 08:23 AM
Posted 11 January 2011 - 01:55 PM
From the multiple peaks it sounds like you may have contaminated some of your cultures with more than one plasmid, some of which are the correct sequence (or at least contain the binding sites for the primers). You may want to try cloning again, or checking to make sure that you are being careful with you PCR products so that these don't contaminate the plasmid stocks.
Posted 11 January 2011 - 03:44 PM
Posted 12 January 2011 - 12:50 AM
Posted 12 January 2011 - 09:28 AM
Posted 13 January 2011 - 09:46 AM
Please check your sequencer. I suspect your sequencer might have some problem, maybe too long no maintenance?
I had an incident where I sent my sequences to a commercial company for sequencing, all my result was ok and clear except there was one sequence got mixed with foreign DNA product, and is appear ~400bp longer (which is a pig DNA after I had blast it), my work never deal with any DNA other than bacteria, and I had verified my sequence before I sent it so I'm confident the problem is not with my side... I suspect the probe in the sequencer might be not well cleaned...
just my 2 cents.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 17 January 2011 - 03:57 AM
Posted 30 January 2013 - 09:59 AM