2 replies to this topic

[sihyunie]

I'm trying to clone in a 500 bp insert into a modified pUC19 plasmid (I removed the lac operon and put in new promoter and MCS) using XhoI and XbaI.
I gel purified both the insert and plasmid after enzyme digest and checked them both after to make sure that they are all fine.
After ligation using Roche quick ligation kit, I transformed and got tons of colonies.
The problem occurred when I checked few colonies.
I digested miniprep products with XhoI and XbaI, expecting to see 500bp band along with 2300bp band. To my surprise, none of the colonies had 500bp band (they had 2300bp band).
I checked both XhoI and XbaI for enzymatic activity.
I even tried removing phosphates from the digested plasmid just as an added measure, but still no positive colony.
I can't really figure out why this simple cloning is giving me an issue.

[rkay447]

sihyunie, on Apr 9 2010, 12:12 PM, said:

I'm trying to clone in a 500 bp insert into a modified pUC19 plasmid (I removed the lac operon and put in new promoter and MCS) using XhoI and XbaI.
I gel purified both the insert and plasmid after enzyme digest and checked them both after to make sure that they are all fine.
After ligation using Roche quick ligation kit, I transformed and got tons of colonies.
The problem occurred when I checked few colonies.
I digested miniprep products with XhoI and XbaI, expecting to see 500bp band along with 2300bp band. To my surprise, none of the colonies had 500bp band (they had 2300bp band).
I checked both XhoI and XbaI for enzymatic activity.
I even tried removing phosphates from the digested plasmid just as an added measure, but still no positive colony.
I can't really figure out why this simple cloning is giving me an issue.

Are you getting colonies on the vector control plate?  How much DNA (insert:vector) are you putting in the reaction?  The problem right now is that you are getting lots of colonies without the insert which you are going to have to fix or screen tons of colonies.  Either you have uncut plasmid present or your phosphatase reaction wasn't efficient and you are getting religation.  What are your ligation conditions?  Sometimes the quick ligation just doesn't work and I have to use T4 ligase.  I do the ligation for a couple hours, transform 4ul and then let the reaction sit on my bench overnight.  If I don't get a positive clone (by PCR screen) I retransform.  Sometimes I only get the product after the overnight.

[pDNA]

XbaI is methylation sensitive ...so dependent on your insert there is the possibility that the Xba-site gets methylated and the enzyme is no longer able to cut. Have you checked that possibility?

Regards,
p