2 replies to this topic

[helloppal]

I am trying to create an insert for deletion of a gene using homologous recombination. For this I amplified the following
1st product : 3 nts-XhoI site-5' flanking region-10 nts-AatII site-4 nts
2nd product : 10 nts-AatII site-4 nts-3' flanking region-XbaI site-3 nts

I then digested 1st product with XhoI and AatII, 2nd product with AatII and XbaI and the vector (pJQ200KS) with XhoI and XbaI.

This was followed by clean up and double ligation for 15 minutes.

I then transformed them in E. coli WM3064 and did colony PCR to verify the inserts.

But, after I grow the positive colonies in media followed by plasmid preps - the insert seems to disappear (No bands from insert PCR verification and vector only bands when digested by XhoI, XhoI+XbaI, XhoI+XbaI+AatII)

Any suggestions?

[ranvi]

why dont you do the plasmid prep and do the digestion instead of colony pcr may be that will work

[christy]

helloppal, on 03 September 2010 - 01:18 PM, said:

I am trying to create an insert for deletion of a gene using homologous recombination. For this I amplified the following
1st product : 3 nts-XhoI site-5' flanking region-10 nts-AatII site-4 nts
2nd product : 10 nts-AatII site-4 nts-3' flanking region-XbaI site-3 nts

I then digested 1st product with XhoI and AatII, 2nd product with AatII and XbaI and the vector (pJQ200KS) with XhoI and XbaI.

This was followed by clean up and double ligation for 15 minutes.

I then transformed them in E. coli WM3064 and did colony PCR to verify the inserts.

But, after I grow the positive colonies in media followed by plasmid preps - the insert seems to disappear (No bands from insert PCR verification and vector only bands when digested by XhoI, XhoI+XbaI, XhoI+XbaI+AatII)

Any suggestions?

You could have design primers for the overlapping of the two fragments.