checking for inserts with colony PCR
Posted 30 August 2004 - 02:41 PM
I am trying to clone a PCR fragment into a vector. I get plenty of colonies. When I check for inserts by amplifying the plasmid DNA from the transformed E. coli with primers from the vector which flank the insertion point, I get a product of the expected size. However, when I sequence the plasmid DNA (again using vector primers as sequencing primers) all I get is vector sequence. My inserts are 1.5-1.8 kb; the vector is ~1.8 kb. Could my PCR primers be amplifying the vector itself somehow? I thought that wasn't possible. Or is something else likely to be happening?
The vector is an extremely low copy number vector, so I don't have enough DNa to do restriction digest.
Also, does anyone have suggestions for cloning PCR products from genomic DNA containing introns? These fragments are being extremely difficult to clone - I've tried several kits, TA, blunt, with and without phosphorylation on the primers.
Posted 30 August 2004 - 03:43 PM
I've had this problem before, with false positives from colony PCR. Do you get a weak ot strong product in your PCR? Sometimes you might have left-over unincorporated DNA in your prep, so that the PCR gives you a positive, even though your vector doesn't have inserts. Do you run a control ligation plate with vector only, no insert? Do you get colonies there? In that case you'll have to modify your cloning procedure.
Posted 01 September 2004 - 08:39 AM
Posted 01 September 2004 - 03:28 PM
are your neds compatible after digesting with RE? Otherwise you shouldn't be getting ANY self-ligated vector.
If they are compatibel- then phosphorylate, and again- no self-anneled vector
Posted 02 September 2004 - 08:54 AM
Posted 02 September 2004 - 01:32 PM
Are you using the low copy (LC) version of the pSmart vectors? If so why no use the high copy (HC)? Have you tried the Invitrogen T/A or blunt TOPO kits? My guess is they will be better than the CloneSmart kit from Lucigen.
What do you mean you don't know of a vector for sequencing that is not linear? Why does that matter? Why not use any vector?
Posted 03 September 2004 - 08:05 AM
I amplify my inserts using Phusion polymerase from Finnzyme. It produces blunt-ended fragments. I phosphorylate my primers before PCR with PNK. After PCR I gel-purify the PCR product before ligation. This is for the CloneSmart kits. For T/A cloning I use the unphosphorylated version of the primers, and after gel-purifying the PCR product I add A overhangs with Taq, then immediatly do the ligation.