useing a uniqeu enzyme for both insert and vector digestion
Posted 05 October 2011 - 03:04 AM
here is the situation:
I am trying to sub clone a fragment from pTZ-57R/T plasmid into another one.
1- the plasmid that contains the insert fragment has been digested with SalI enzyme in order to digest out the insert fragment.
2- after the completion of digestion process, the desired band has been purified from the agarose gel.
3- the ultimate vector has been digested with the same enzyme and after the completion of the digestion process and checking the lineariziation of the vector on agarose gel, it has been treated with alkaline phosphatase and then purified.
4- both vector and insert have been incubated in 37 centigrade degrees for 15 mins. prior to ligation reaction.
5- ligation reactions were set up with different I:V ratios( 2:1, 3:1, 6:1) and incubated in different situations( 16 centigrade degrees or 4 centigrade degrees) for an overnight period of time.
6- the following day, chemically competent bacteria has been transformed with these ligation reactions and after an overnight incubation in 37 centigrade degrees, no colony was seen on the plates.
how can the ligation efficiency be induced in such situations?( when the only option is the use of an unique enzyme for both vector and insert)
all the best
Posted 05 October 2011 - 05:52 AM
Let us know and we'll try to help.
Best of Luck.
Posted 07 October 2011 - 01:47 AM
1- apo.150 ng( I have tried 50 ng as well) vector, 1-3 microlit
2- relative ng of insert to keep it within aforementioned molar ratios with Vector , 5 or more microlit
* both vector anb insert are eluted in distilled water.
3- 10/5 X T4ligase buffer, 2/4 microlit( the stock buffer has been previously dispersed in order to avoid repetitive freez/thaw, DTT smell is dominant and I avoid vortexing the buffer)
4- T4 ligase, 1/5 units/microlit. 1- 2 microlit.
5- d.W up to 20 microlit.
2- yes, both vector and Insert will be cooled either on Ice or left to reach the room temperature before proceeding to the next step.
3- lab-made chemically competent Ecoli, I have used different strains: DH5alpha, JM110, GM2163
thank you in advance
Posted 07 October 2011 - 02:33 AM
How do you transform your cells?
Posted 07 October 2011 - 02:46 AM
the method I use for transformation is sambrook's protocol:
1- mix the ligation reaction with 200 microlit of competent bacteria and incubate on ice for 30 min
2- incubate in 42 centigrade degrees for 90 sec.
3- transfer on ice immediately incubate for 1-2 min
4- add 700 microlit of prewarmed LB, incubate in 37 centigrade degrees for 60 min
5- plate on LBagar Amp posotive, incubate overnight.
6- check for colonies
I dont use a ligation control.
Posted 07 October 2011 - 05:55 AM
If that isn't your problem, then I am stumped. I'll keep thinking about it, but definitely let us know if something works.
Best of Luck.
Posted 08 October 2011 - 03:15 AM
Posted 08 October 2011 - 03:44 AM
I would suggest strongly that you test your cells for competence. Ligation problems, in my experience, are almost always problems in the DNA being used, or (more commonly) low competence cells. If you have good DNA and good cells, the ligation is rarely a problem. Ligation buffer can, with many freeze/thaw cycles or age cause difficulties.
Posted 08 October 2011 - 04:23 AM
Posted 08 October 2011 - 04:51 AM
Posted 08 October 2011 - 05:19 AM
thank you indeed for the elaborated explanation, may I ask what method do you use to prepare chemically competent Ecoli? the standard CaCl2 method? I use MOPS buffer and compared to the CaCl2, looks like it works better.
Posted 08 October 2011 - 05:53 AM
Posted 08 October 2011 - 07:40 AM
Lastly, I will strongly recommend that you stop screening with colony PCR and start mini-prepping plasmid and screening that way. Colony PCR is a great quick and dirty way to screen clones when things are working, but once you start having so many problems, you want to be using samples that you can depend on. This includes using millipore water (or PCR water if you buy it). There can be all kinds of enzyme inhibitors present in crude DNA preps, colony PCRs, and non-standard water sources, and right now, you need to eliminate as many questions as possible.
Best of Luck.
Posted 22 October 2011 - 09:29 AM
thank you for the support and tips, IT FINALLY WORKED.