6 replies to this topic

[turtle]

Hi.
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.
Thanks,
Rebecca

[kant0008]

Hi,
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.

[turtle]

I get strong positives with the PCR, and I get significantly fewer colonies in the no-insert control. So if the problem is with the cloning method it is in the insert-vector ligation itself.

[kant0008]

Hi,
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

[turtle]

I'm doing blunt cloning, so no restriction enzyme digestion. I could redesign my primers to add RE sites to my insert, but I don't know of a source of vectors for sequencing (rather than expression) that aren't already linearized. I've been using the CloneSmart line from Stratagene. I've also tried T/A cloning using Invitrogen's sequencing vectors and pDrive from Qiagen.

[wirly]

What PCR enzyme(s) have you used and in what combination with phosphorylation state and cloning kits (T/A and blunt)?

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?

[turtle]

I have tried a variety of high-copy kits, and had generally poor results. I am trying to clone genomic DNA from plants - complete with introns, which are very A/T rich. My experience has been that the bacteria have a tendency to excise my inserts from the plasmids during replication. I am reluctant to use expression vectors, because I expect that having the bacteria trying to express the plant genes will only make matters worse. I switched to the LC-kan kit from Lucigen because it is supposed to be the most tolerant of A/T rich and toxic inserts.

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.
-Rebecca