an annoying cloning - (Apr/02/2009 )

My target gene is 2.3 kb, use primers (Forward primer adds NheI restriction site before) to amplify target gene, purify it with kits, digest it and vector with NheI and PstI restriction enzymes, all these steps were checked on the gel.
Ligate them (insert 2.3 kb, vector 2.8 kb, insert: vector=3:1) overnight at 4 C, heat shock transformation, then pour transformant reaction on LB.
Check colonies for sequencing, but the results are all not correct, till now I have done many times, but still no advance.

Any suggestion is welcome!
Many thanks in advance!

Biogareth

do you have additional bases to the restriction enzymes while designing primers.
atleast PstI enzyme requires three additional bases after the restriction site.
http://www.promega.com/guides/re_guide/chaptwo/2_6.htm#pp
good luck

metta on Apr 2 2009, 07:34 AM said:

Thanks!
I added additional bases before restriction site followed NEB.

Do you clean your vector and insert after restriction?
PstI can't be completely inactivated, so this step would be necessary.

mastermi on Apr 2 2009, 08:26 AM said:

I used QIAquick gel extraction kit to purify them after digestion

How was the background (vector + ligase)? If the background is high, you probably need to dephosphorylate you vector even you have two enzymes for digestion

I have found this helpful:

Digest the vector, dephosphorylate and precipitate the reaction, reconstitute in water and use directly for ligation. Somehow agarose inhibits ligations and this works.

Best,
TC

Actually just out of curiosity right, when you say the sequence is wrong issit like a completely different gene that you got or what?

That seems to be my main concern as of now. could it be some flaws in the primer design?

If you use retro or lenti vectors, you might get recombination by using the wrong competent cells........

If you use pcDNA and other expression vectors, you might (over)express a poison proten (even without inducer) and becteria may not like it.............

There are many possibilities that you won't get you clones. So, you might want to describe your problem in more detail.

Thank you all for advice!
Because I think the problem is that PCR products with insert cannot be cut well by restriction enzymes, later PCR products with insert was cloned into pGEM-T first, and then white-blue screen, I already got the plasmid with insert (checking by sequencing), afterwards cut the plasmid with NheI and PstI, the new plasmid has one NheI site and two PstI sites, but I think it can not be the problem to get the right insert, I already checked it on the gel.
As far as the digestion of vector was considered, I also check on the gel, it should be no problem.
After that, I do the ligation with different ratio of insert to vector (2:1. 3:1, 9:1), both overnight 4 C and room temper for 2 hours, but I just got one colony on the plate, no colony on control (without insert). When I checked this colony by colony PCR, there is still nothing, all the bands at the bottom of gel! It's so pity!
I already worked on that for 3 months! How come?

Many thanks in advance!

Biograreth