cloning problem - (Jul/23/2007 )
Hipeople
please give me a solution to overcome the problem I face in my cloning expt., I use binary vector pBI121 to clone my 800 bp insert. Its a directional cloning. The vector is linearized with double digestion of R.E and then vector is eluted from gel. So is my insert. I use 3:1 insert : vec ratio for ligation in a 16ul ligation mix which constitutes 100U of T4DNA ligase. After transformation, I was able to see five colonies in my test plate. Out of which three were white in colour, and I don believe its E.coli. In my negative control (with antibiotic)plate (plated with E.coli host cells) also I was able to seee three such colonies. I tried it two times. the result is the same. The competent cell stock is used by other lab members too but with different plasmid. Its working fine with others. so I dont think any problem with competent cell. can anyone suggest where else the problem can occur? your suggestions are highly needed. thank you.
your insert or your vector or both may not have been cut properly. are you sure both R.E.s are working well? have you had a self-ligation control? you may ligate your double cut vector and transform it to see whether it self-ligates or not. tell us more about your experiment; how long do you cut your DNAs, for example?
you may try some other insert:vector ratios like 1:1, 1:5, 1:8 . at what temperature are you performing your ligation reaction? try 4C ON incubation. you may add PEG 6000, 15% final conc., into your ligation reaction. PEG makes the volume compact so your vector and insert have a better chance to find eachother.
your vector is huge, there might also be a problem in transformation. do you use chemical transformation or electroporation?
And finally, have you checked your white colonies in your test plate, are they empty vector? why do you think they are not E.coli?
you may try some other insert:vector ratios like 1:1, 1:5, 1:8 . at what temperature are you performing your ligation reaction? try 4C ON incubation. you may add PEG 6000, 15% final conc., into your ligation reaction. PEG makes the volume compact so your vector and insert have a better chance to find eachother.
your vector is huge, there might also be a problem in transformation. do you use chemical transformation or electroporation?
And finally, have you checked your white colonies in your test plate, are they empty vector? why do you think they are not E.coli?
Dear dodosko
I'm sorry for not giving u the exact details of restriction digestion. I use BamHI and SacI to linearize my vector and use the same to release the insert from pGEMT clone. I follow 3 hours digestion at 37 deg. This time of incubation had worked well for me. When I digest my vector it should release 1.8 kbps. I am able to see that insert release from the vec. Regarding my insert it also aligns at the correct size. so I hope there's no problem with my restriction digestion or enzymes. I suspect the problem to be with ligation.
I dint have a self-ligation control. The ligation reaction is incubated at 16 deg o/n. I use CaCl2 mediated transformation. I kept a control of my vector alone in transformation. It had no problem giving very good no. of colonies, which are E.coli not the white colony I got in test plate.
I checked those white colonies on the selection plate, then tried to isolate plasmid from them and did colony pcr. The colony has grown on selection plate but colony PCR and plasmid isolation results were negative. I said they were not E.coli since the colour of the colony was bright white and were not translucent as E.coli colony.
Hope this explanation would help you find me a solution. Thanks in advance.
it may b ligation problem keep it in PCR at 4 c overnight
Overnight ligation works the best.
5 colonies are way too little for transformation efficiency.
Since it is not your competent cells problem, it lies on your ligated product.
people I've tried 16 deg and 4 deg overnight incubation for my ligation reaction, but in vain. Can anyone suggest me some tricks for transforming a bigger plasmid in E.coli. such as some changes in my regular heat-shock transformation protocols.
also tell me how much units of ligase works better for sticky end cloning?
waiting for your help.
I think your transformation efficiency is low. CaCl2 transformation is not very good for ligations, but fine for re-transforming plasmid DNA. Make or buy more competent cells. This is my favorite protocol:
http://openwetware.org/wiki/TOP10_chemically_competent_cells
For T4 ligase, you need only very small amounts for sticky end ligations. For a 16 ul reaction, you should use the smallest amount you can reliably pipet, say 0.2 ul, or dilute the ligase 10x (see the web site for how to do this).