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Problems subcloning with the pL452 recombineering plasmid - (May/30/2008 )

We are trying to modify the pL452 recombineering vector (by insertion of a 1 Kb fragment) with no success and would love to hear any suggestions. We believe the problem lies with the vector since we cannot even re-circularize the vector after a single cut with 7 different REs (no phosphatase used). The vector cuts very well in all cases and our DNA yield after gel purification is consistently around 30 ug/ml. The vector is of high purity and has been propagated in multiple strains of conventional bacteria but this doesn't solve the problem. We don't have this problem with any of the other multiple vectors used in the lab and we know from other cloning experiments that all the other reagents: REs, ligase, competent cells etc. are working appropriately. We have phenol/CHCl3 extracted the DNA fragments and we have even tried re-phosphorylating the DNA ends with T4 PNK to no avail in either case. In my 15 years of cloning I have never come across such a problem but am hoping someone out there has.
Thanks!

-Heels-

QUOTE (Heels @ May 30 2008, 05:11 AM)
We are trying to modify the pL452 recombineering vector (by insertion of a 1 Kb fragment) with no success and would love to hear any suggestions. We believe the problem lies with the vector since we cannot even re-circularize the vector after a single cut with 7 different REs (no phosphatase used). The vector cuts very well in all cases and our DNA yield after gel purification is consistently around 30 ug/ml. The vector is of high purity and has been propagated in multiple strains of conventional bacteria but this doesn't solve the problem. We don't have this problem with any of the other multiple vectors used in the lab and we know from other cloning experiments that all the other reagents: REs, ligase, competent cells etc. are working appropriately. We have phenol/CHCl3 extracted the DNA fragments and we have even tried re-phosphorylating the DNA ends with T4 PNK to no avail in either case. In my 15 years of cloning I have never come across such a problem but am hoping someone out there has.
Thanks!


1. As you are an experienced person, and as vector can not even re-circularize, I suspect there is some problem in Ori of replication. Given that your ligation & etc techniques are good. PL452 has worked great with me. I have made about a dozen constructs using it in the past.

2. You may as well ask the NCI recombineering people to send you PL452. They are quick, you will get it in 'bout three days. Free, as you may know it.

..

-cellcounter-

QUOTE (cellcounter @ May 30 2008, 10:22 AM)
QUOTE (Heels @ May 30 2008, 05:11 AM)
We are trying to modify the pL452 recombineering vector (by insertion of a 1 Kb fragment) with no success and would love to hear any suggestions. We believe the problem lies with the vector since we cannot even re-circularize the vector after a single cut with 7 different REs (no phosphatase used). The vector cuts very well in all cases and our DNA yield after gel purification is consistently around 30 ug/ml. The vector is of high purity and has been propagated in multiple strains of conventional bacteria but this doesn't solve the problem. We don't have this problem with any of the other multiple vectors used in the lab and we know from other cloning experiments that all the other reagents: REs, ligase, competent cells etc. are working appropriately. We have phenol/CHCl3 extracted the DNA fragments and we have even tried re-phosphorylating the DNA ends with T4 PNK to no avail in either case. In my 15 years of cloning I have never come across such a problem but am hoping someone out there has.
Thanks!


1. As you are an experienced person, and as vector can not even re-circularize, I suspect there is some problem in Ori of replication. Given that your ligation & etc techniques are good. PL452 has worked great with me. I have made about a dozen constructs using it in the past.

2. You may as well ask the NCI recombineering people to send you PL452. They are quick, you will get it in 'bout three days. Free, as you may know it.

..


Many thanks for your suggestion, it does make a lot of sense. We are in the process of growing up a new batch of pL452.

-Heels-