4 replies to this topic

[Jaaar]

Fellow, I got a problem with the blunt end ligation of the pETBlue-1 vector from Novagen with zero positive colony after PCR screening

The procedure I did for the cloning was:

Insert
1. Clone the fragments (~600bp) using Platinum pfx Taq (which is claimed to have no dA addition)
2. Purify the fragments by methods previously preferred.
3. Phosphorylate the fragments using T4 polynucleotide Kinase from NEB under T4 DNA ligation Buffer

Vector
1. Digest pETBlue-1 with EcoRV (vector size ~3.5 kbp)
2. Dephosphorylate the linearized vector using CIP
3. Purify the fragments by methods previously preferred.

Ligation
1. Ligate the dephosphorylated vector and phosphorylated insert using T4 DNA Ligase from NEB with Insert:Vector = 3 : 1 at 16 ℃ ON
2. Transform the ligation products to E.coli and do Blue-white screening.
3. Screen the white colonies using GoTaq PCR

The result was really disappointing because no positive clones obtained; meanwhile, there was no colony in the negative control (vector only ligation). I tried to further raise these colonies to extract the plasmid for RE digestion. The resulting pattern was all matched with the vector.

Any solutions ?

[pDNA]

if you got false positive clones on your plates and no clones on your vector only ligation this would mean you have a carry over of plasmid DNA from the Insert?
...or did i got i wrong and you have no colonies at all?

you can try a ratio of 5:1 at room temperatur for at least 2h.
before ligation check vector and insert on a gel ...to reasure you use the right quantities for your calculations.

do all controls necessary and check self-ligation of vector and insert on a gel.
something must be wrong with eather vector or insert ...so you'll have to troubleshoot.

Regards,
p

[Jaaar]

pDNA, on 17 October 2011 - 07:18 AM, said:

if you got false positive clones on your plates and no clones on your vector only ligation this would mean you have a carry over of plasmid DNA from the Insert?
...or did i got i wrong and you have no colonies at all?

you can try a ratio of 5:1 at room temperatur for at least 2h.
before ligation check vector and insert on a gel ...to reasure you use the right quantities for your calculations.

do all controls necessary and check self-ligation of vector and insert on a gel.
something must be wrong with eather vector or insert ...so you'll have to troubleshoot.

Regards,
p

Thank you for your suggestion, p!

The question is that the insert was directly cloned from another plasmid with totally different features comparing to pETBlue-1, e.g. KanR, no LacZ. The possibility of carry-over of pETBlue-1 from insert should be eliminated.

I did the ligation at 16℃ overnight which i suppose the reaction time is way-more sufficient. I came across a thought that may be it is due to the toxicity of the insert to the microbes once expressed.

[pDNA]

okay! ...so no carry over from the insert.
you have a clue if your false positive contain any insert or is it just empty vector?

are you doing blue/white screening?
...and what E. coli strain do you use for cloning?
as long as you do not use a DE3 lysogen (anyway not recommended for cloning!!!) there should be no translation from the T7 promoter.
The only problem could be the promoter of the lacZ ...since you do blunt end cloning and not directional cloning it could be that the insert gets expressed when it is in direction of the lacZ promoter.

Is it possible to do directional cloning?

Regards,
p

[Jaaar]

I did restriction mapping that ended up indicating the false positives were carrying the empty vector.

The E.coli used was DH5alpha, very efficient for my previous cloning projects.

My goal is to try out the pET expression system for my target protein. I have only the pETBlue-1 vector from the Perfectly Blunt Cloning Kit on hand, which is said to achieve optimal expression when inserted bluntly in frame. I know that directional cloning is much more easier to get it done...

Thanks again, p!