ligation and transformation

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scchaudhary
scchaudhary's picture
ligation and transformation

Dear all !
I have hard time in ligation . i cut PKH3 vector and  avian Rsk1 with single  as well as double cut simultaneously with BamH1 and Hpa1 .when checked  bands were expected.but when i used to ligate vectorPKH3(4.2kb) with insert RSK1(2.8kb) and PKH3(4.2KB) with PKH3( 600bp)  together as +ve control but i couldnot get any colony after transformation .my competent cell and ampicilline plate has no problem as i checked with another plasmid DNA. i took 1:3 vector and insert ratio.still i didnot succeed in this experiment. i did as you suggested me previously .but unfortunately no colony appeared. therefore , plz suggest me again so that i can overcome my problem.
By the way, how can i find molar ratio of vector PKH3(4.2KBP)  and avian RSK1( 2.8KBP) ? i found vector concentration 220ng/microliter  and 75 ng/microliter of insert RSK1 after measuring.
how can i know whther my ligase and ligase bfr isworking or not , is there any method to check  or we we have to check with  new bfr and ligase?
Iam waiting your kind cooperation.
I hope you will writr me very very soon .
Thanking you again

RLS
RLS's picture
 Did you gel purify the

 Did you gel purify the vector? Did you phosphatase treat the vector? These are both steps I would include, but it doesn't seem to be the problem that your vector is ligating back to itself since there are no colonies, which is good. 

Secondly, how was your insert generated? By PCR with the restriction sites included at the ends of the primers? If so, did you make sure to add enough bases at the end of each one so that the restriction enzymes could cut effectively? If  you are unsure, check the New England Biolabs website; it tells you how many bases you need to cut most efficiently. Whenever cloning inserts, if your lab has the money, I always suggest cloning the insert into the TA vector (kit by Invitrogen; make sure you use fresh PCR product immediately after PCR is finished if you use the one with A overhangs. Then you can cut your vector out, knowing for certain that you have the correct restriction sites. Gel purify the insert away from the vector at this point. 

As far as your ligase and ligase buffer: if your ligase buffer is possibly old, the DTT may be degraded. Either make some up yourself: 10X buffer: 500 mM Tris-Cl, pH 7.6, 100 mM MgCl2, 100 mM dithiothreitol (DTT), and 500 ug/ml BSA. To test, cut your vector with one enzyme, do not dephosphorylate, gel purify to make sure it is cut, and re-ligate it. You should definitely get colonies upon transformation. 

If you have tried a 1:3 ratio, maybe try a couple of other ratios, once you are certain your ligase is actually active, such as 1:5. What are your conditions for ligation. I typically use 16 degrees C overnight. 

Hope this helps!

Lynn

Abhishek Singh
Abhishek Singh's picture
as much as i understand  for

as much as i understand  for ligase u can do cut vector and genome and take half of sample  for ligation. and then check whether lgated sample and not ligated one is showing difference in electrophoressis band pattern.

scchaudhary
scchaudhary's picture
Hi

Hi
Thanks  alot all of you who suggested me for my problem.
However my  objective is to just check the ligatin process.by the way  i didnot perform the phosphatase treatment , so plz can you help me how to do the phosphatase treatment  and how can we dephosphorylate the  cut vector , if we need.
how can we find the molar ratio of vector and insert.

thanking you again