Protocol Online logo
Top : Forum Archives: : Molecular Biology

Please helpI need a failsafe cloning method - (Feb/26/2007 )

Same old problem with a twist.
I have been trying to clone a 500bp insert into pGL4 23 vector. Insert cut with Nhe1 and Spe1 and vector with Nhe1.
Have tried various approaches but no postive colonies.
Done number of control experiments....ligase is working....enzymes cutting....cells viable etc but can't find out what the problem is.
The twist is I have been told I have got one month to sort this out but my I am not allowed to test different vector: insert ratios, I am also not allowed to test more than 6 colonies per plate as my PI does not see value of this....too expensive....my PI also doesn't believe in colony PCR, or trouble shooting in any shape or form.
Help

-lugworm-

Have you tried dephosphorylation of your vector? Then you prevent selfligation of the vector. Weird PI you have, colony PCR is a very quick method to screen your clones on the plate, and you know soon enough if you have the correct clone. I do it often when my ratio is bad between my control plate and my vector-insert plate.

-brigitta-

Ya, a new PI would be your best bet.

-wbla3335-

Thanks for that....I agree totally ....re PI. I have dephosphorylated vector. No insert control or CIP treated vector gave no transformants. My fear is that I could be missing positives because I am not allowed to do a full screen. I was also shouted at for setting up an experiment to test for optimal insert:vector. I needed that whinge

-lugworm-

No positive colonies? What exactly do you mean? You checked your colonies by what method? Or did you have no colonies on your supposed to be positive plate? What do you see on your negative control plates?
What methods have you used to purify your vector/insert?
What's the origin of your insert? PCR product or cut from a plasmid?

-vairus-

How long did u digest ur fragments? try to reduce digestion time. Couldnt you try different sites?

-scolix-

PI is an ass, for a start.
Apart from that, can you use another enzyme apart from Nhe1? It sounds like its a bit difficult to work with - http://www.neb.com/nebecomm/products/faqproductR0131.asp#846
It's a commercial vector, there should be a MCS with heaps of restriction sites to choose from. Where does the insert come from? Has it been cut out of another vector?
The trouble is that if I understand you right, your insert can go in either forwards or backwards, and your vector could also re-ligate. This means that if you only check 6 colonies, your chances of finding what you want are really very low...

-scrat-

you should explain to your PI that it costs more to pay you to fail at this experiment for a month than it does to screen for positive colonies.

-Eric J-

QUOTE (scrat @ Feb 26 2007, 10:26 PM)
PI is an ass, for a start.
Apart from that, can you use another enzyme apart from Nhe1? It sounds like its a bit difficult to work with - http://www.neb.com/nebecomm/products/faqproductR0131.asp#846
It's a commercial vector, there should be a MCS with heaps of restriction sites to choose from. Where does the insert come from? Has it been cut out of another vector?
The trouble is that if I understand you right, your insert can go in either forwards or backwards, and your vector could also re-ligate. This means that if you only check 6 colonies, your chances of finding what you want are really very low...

I agree with scrat.

Your cut vector can reliagte even though dephosphorylated, maybe efficiently. Check more than 6 colonies is qiute necessary , and obviously, that is not expensive as your PI said.

-Hyland-