Problem with subcloning - (Aug/09/2005 )
hi freinds
I am having problem in ligation.I have to subclone a gene in IRES eGFP vector,I digested the gene with Not1 to get the pop out from the transient vector.I digested the IRES eGFP vector with sma1.Then I used T4 DNA Polymerase for creating the blunt end.After this ,I column purified and used phosphatases.Then I did the ligation .But I didn't get any colonies.Can anyone help me out and tell me what are the precuations which I should take while performing cloning.
thanx 
You haven't really given us enough information to help you. What controls did you do? What were the results of the controls?
One tip is don't use T4 DNA polymerase to create the blunt end, but use klenow instead. T4 3'5 exonuclease domain is very active making it hard to get true blunt ends.
Daniel
Longer automated DNA sequencing reads
One tip is don't use T4 DNA polymerase to create the blunt end, but use klenow instead. T4 3'5 exonuclease domain is very active making it hard to get true blunt ends.
Daniel
Longer automated DNA sequencing reads
I will take the suggstion and use Klenow now.In ligation I didn't put any control.But during transformation I used the positive control( uncut vector).I got good transformation effeciency.What should be the total volume of ligation mixture.I used 20 microlitre.How long can be the digested and column purified DNA stored .Please inform me
thanx
You will need to do more controls to work out what is going wrong. Blunt end cloning can be tricky as there are so many steps that all have to work. Basically make a positive control for every enzymatic step.
20µl volume should be fine. How long the DNA can be stored depends on the conditions (temp, buffer, if nucleases are present, etc) so it is difficult to say.
Finally, are you using arctic shrimp phosphatase? Calf alkaline phosphatase can be very difficult to inactivate and often carries through and de-phophorylates your insert during the ligation (no clones).
Daniel
DNA sequencing reagents