Types of igation controls (View forum version)



KhaleesiDany

Posted 25 May 2015 - 09:26 AM

Greetings people

 

Im doing a ligation of a 6kb vector and 1kb insert. If my vector is cut with two different enzymes (they produce sticky ends) would i still need to do dephosphorylation?

Also which controls do i add for ligation. I know positive control would be a cut vector to see if ligase works. My negative control is it vector DNA and ligase but no insert? how is it different from Vector DNA no ligase and no insert? or should i do both negative controls

 

thanks in advance 


bob1

Posted 25 May 2015 - 12:43 PM

Vector, no insert, no ligase tells you if your cutting has been efficient - i.e. that you don't have uncut (or self-ligated) plasmid in your ligation.

 

With different sticky ends you shouldn't need to dephosphorylate.


phage434

Posted 26 May 2015 - 02:22 AM

In my opinion, the most important one is to test the competency of your cells.  Poor competent cells are the most likely failure.  Test by taking a common plasmid (pUC19 for example) and diluting to 50 pg/ul. Some cells come with this control DNA. Transform 1 ul and calculate the colony forming units per microgram of plasmid for your cells. You should have 10**8 or so for cloning. 10**7 is barely sufficient and cause for worry.


KhaleesiDany

Posted 26 May 2015 - 05:39 AM

Vector, no insert, no ligase tells you if your cutting has been efficient - i.e. that you don't have uncut (or self-ligated) plasmid in your ligation.

 

With different sticky ends you shouldn't need to dephosphorylate.

Do you suggest ligation overnight at 16 or 4 degrees Celsius  ? 


phage434

Posted 26 May 2015 - 10:04 AM

If your sticky end ligation is going to work, it will work with a 10 minute room temperature ligation on the bench. Doing it overnight is a good excuse if you need to go home, but won't usually solve your problems.


KhaleesiDany

Posted 27 May 2015 - 12:22 AM

If your sticky end ligation is going to work, it will work with a 10 minute room temperature ligation on the bench. Doing it overnight is a good excuse if you need to go home, but won't usually solve your problems.

 im using a t4 DNA ligase kit. It says 5 weis U per microlitre on the bottle.. and the protocol says use 1 weiss U does that mean I should use 0.2 microlitres? I have always been just using 1 microlitre and ligations have been working but recently failing could it be that 1 microlitre of t4 is too much for the reaction?


phage434

Posted 27 May 2015 - 12:04 PM

It is an ENZYME not a reaction component. This means you do not need to calculate molar amounts -- more will simply work faster. Use a microliter and don't overthink it.