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cloning very long fragments into vectors using T/A cloning method,


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#1 mahsa

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Posted 28 September 2010 - 09:26 AM

multiway ligation is gonna be one of the major techniques i have to deal with all the way through my thesis, and they look like , if i may say, pretty hard ones. first one is ligation that has to be done to clone a 7377 bp fragment into a 3000 bp vector. to increase the efficiency I have treated the PCR product with taq DNA polymerase to make sure that the majority of fragments have got the A in their end. then the reaction mix has been incubated in different conditions:
1- 4 c for an overnight period
2- 22.5 c for half an hour and then 16 c for an overnight period
at the end of incubation I have tried both deactivating the ligase by keeping the reaction mix in 65 c for 20 min and not going through the deactivation step.
finally i have transformed the competent bacteria with the transformation reaction.
the molar ratios I have used are 1:1 and 1:2.
i have screened the colonies by both blue/white screening and antibiotic resistance. then i have performed colony pcr for unlimited number of colonies( occasionally i have checked blue colonies in addition to white colonies). I have had no luck till now, even absolutely white colonies don't have the insert as they are suppose to.
Are there any other ways of increasing the efficiency of ligation, because obviously this step is the hold back here.
tnx
PS: I have tried different vectors for T/A cloning step

#2 HomeBrew

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Posted 28 September 2010 - 11:44 AM

Two thoughts:

First, must you TA clone? Can you add restriction sites to your primers and clone the amplicons after RE digestion? And secondly, how are you doing your colony PCRs? There's no way to differentiate between a negative PCR reaction because there's no insert, and a negative PCR because your colony PCR failed. Did you check any of your "absolutely white colonies" by restriction digest?

#3 mahsa

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Posted 29 September 2010 - 03:40 AM

Two thoughts:

First, must you TA clone? Can you add restriction sites to your primers and clone the amplicons after RE digestion? And secondly, how are you doing your colony PCRs? There's no way to differentiate between a negative PCR reaction because there's no insert, and a negative PCR because your colony PCR failed. Did you check any of your "absolutely white colonies" by restriction digest?



actually there are specific restriction sites on both forward and revers primers, but they are not compatible with the vector which I am going to use for the bacterial part. they are going to serve for the eukaryotic part to clone the fragment after being modified in a eukaryotic vector.
as a matter of fact I have tried the digestion-ligation strategy once, just out of curiosity and with another vector other than the one I have to use, and it didn't work either.
for each round of colony PCR I check the PCR process by both positive and negative controls. would you kindly find a sample in attached file. lane 2 in both upper and lower row are the negative control and the first lane in both rows are the positive controls.

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