Help!!! - Unsure of problem (Oct/07/2009 )

Hi

I need help and advice regarding my cloning technique, quite simply because I am not yielding anything.

I am cloning 4 constructs, each of which is being cloned into two pET vectors (pET17b and pET22b)
I am using NdeI and XhoI (NEB). My primers account for the necessary overhang needed 5' of the restriction sites.

I PCR using AccuPrime pfx (invitrogen) and yield great quantities of PCR product following gel purification (≥200 ng/μL)

For all my PCR products and Vectors I digest for 2.5 hours using NEB buffer 4 and BSA
My vector is further treated with Antarctic phosphatase for 20mins and deactivated for 5 mins at 65C

From here I gel purify double digested vector by gel purification and PCR product by EtOH precipitation (This allows me to retain high quantities of both PCR product and vector ≥100ng)

Following this I have performed ligation at varying conditions and times
16C for 1 hour
16C for 16 hours
RT (23C) for 1 hour

I have also performed 3:1, 4:1, lots:1 of insert:vector to no success

I know my ligation can work as I have run a positive control with double digested vector + cut insert produces colonies.
My negative control has been run with cleaned up double digested vector with ligase which yielded no colonies

Please help, why am I the only one on my level not getting clones

Pat

-PaddyS-

Likely problem areas:
* UV exposure during gel imaging. Use short exposure to long wave UV or blue light.
* Lack of purification between PCR and cutting, resulting in PCR fill in of the overhangs
* Too high a concentration of DNA in restriction digests, ligations
* Ligation at too high a temperature
* Too much ligation mixture being used for transforming competent cells
* bad transformation efficiency

I wouldn't go near a gel except for diagnostic purposes for this reaction. There is no reason to gel purify your vector after digestion and phosphatase treatment. If your PCR product is good, just purify with a column, digest, and go. NdeI leaves short, easily disrupted overhangs. Ligate at low temperature. You are probably using too much DNA in your ligation. 10-20 ng is enough for both insert and vector.

-phage434-

Thanks

phage434 on Oct 8 2009, 10:31 AM said:

I wouldn't go near a gel except for diagnostic purposes for this reaction. There is no reason to gel purify your vector after digestion and phosphatase treatment. If your PCR product is good, just purify with a column, digest, and go. NdeI leaves short, easily disrupted overhangs. Ligate at low temperature. You are probably using too much DNA in your ligation. 10-20 ng is enough for both insert and vector.

So do you think this would sound reasonable?

Perform PCR and use 2-5uL of this to perform an analytical gel to check meanwhile clean up the PCR using something 'Promega wizard PCR cleanup'

For vector and insert, digest these, phosphatase treat vector

EtOh both of these

Test concentration of both then perform ligation at 4C for 16hr using 20-30ng of vector and appropriate amount of insert

Also, what do you mean by NdeI leaving short easily disrupted overhangs...do you mean it doesnt leave the optimal cut site every time?

Thanks phage434

-PaddyS-