Ligation problem - (Oct/17/2011 )
A couple of questions that might seem obvious but just in case.
1. do you have a transformation control?
2. do you have a ligation control?
If struggling with blunt end, could you not just redesign the primers to include RE and do directed cloning? Your first post with this problem is from over a month ago, I think is time to change strategy. Cloning can be a pain in the b*** but should not take that long to work either 
almost a doctor on Fri Dec 2 11:54:37 2011 said:
A couple of questions that might seem obvious but just in case.
1. do you have a transformation control?
2. do you have a ligation control?
If struggling with blunt end, could you not just redesign the primers to include RE and do directed cloning? Your first post with this problem is from over a month ago, I think is time to change strategy. Cloning can be a pain in the b*** but should not take that long to work either
I have an intact plasmid at 150pg as transformation control and single digested plasmid as ligation control. n both give colonies.
we designed the primers for directed cloning with EcoRI/BamHI (the one month ago post!) which was not working (we put less of the extra bp at the ends - the enzyme was not cutting) hence planned the blunt ligation (instead of designing a new primer)
thought "blunt ligation - its a piece of cake!" STUPID ME! I am new to this cloning business!
anyway thx for the reply! but if u have any suggestion plz post!!! thx in advance!
When you transform 1 ul of your 150 pg/ul DNA, how many colonies do you get? Why are you recovering your cells in LB instead of SOC?
I suspect your problem is in the SAP treatment, but it could also be the kinase reaction. Try SAP treatment for shorter periods (or omit it) and see what happens.
Are you certain that the PCR enzyme in your kit produces blunt ends?
phage434 on Fri Dec 2 21:30:34 2011 said:
When you transform 1 ul of your 150 pg/ul DNA, how many colonies do you get? Why are you recovering your cells in LB instead of SOC?
I suspect your problem is in the SAP treatment, but it could also be the kinase reaction. Try SAP treatment for shorter periods (or omit it) and see what happens.
Are you certain that the PCR enzyme in your kit produces blunt ends?
when I tranasform 150pg DNA, the colonies I get are many (maybe 100-150) but not a lawn as I expected. so there is something wrong with my competent cells. (thx! i did not think of that till now!
) and what is wrong with recovering the cells in LB. I have done recovery in SOC before and I dont see any difference between the 2.what can be the problem with the SAP treatment? (I thought its supposed to help me omit the background. thats good for me right?
) I think my SAP treatment is working fine 'coz there are no colonies on the control plate (vector, no insert ligation) I suspect its the kinase which is not working and hence no colonies on insert+vector plate. Am I thinking right here?
About the PCR... well i dont know if its giving me perfect blunt ends. its an RT-PCR kit from invitrogen with reverse transcriptase and taq polymerase. Taq does not give blunt ends?
For decent efficiency of 10^8 cfu/ug you should be getting about 15000 colonies, so your efficiency is quite low. This alone could be your problem. LB will work for recovery, but it's not standard, and you're looking to make things work as well and as standardly as possible. SAP can not only dephosphorylate, but also trash the ends of DNA, so over digestion will give problems. Less is more. I'd be more concerned about failing to get any colonies in a ligation than in getting zero in the control. We don't know if the kinase is working. Taq does not typically give blunt ends, and could be another cause of your problems.
have you ever tried using PEG in your ligation reaction?
mahrak on Sun Jan 8 15:27:23 2012 said:
have you ever tried using PEG in your ligation reaction?
when ever i do blunt end ligation, i use PEG. n if i am using the quick ligase from NEB, the ligase buffer already has PEG. Why do u ask? I should not be using PEG?