problem with ligation with ClaI - Cloning (Apr/19/2005 )

Hi

I am trying to clone in an insert of approx 700bp into pCs2+ vector using ClaI. However, I have been completely unsuccescful. I believe the ClaI cuts because when i do a double digestion with ClaI and XhoI I get bands of the right size. However after restricting the vector with ClaI and ligating it I dont get any colonies. Both the vector and the insert have been grown in SCS110 strain. I have also tried diffrent molar ratios. The ClaI is from fermentas any suggestions about where I should buy it from?
Can somebody help me with this please?
I have done it a number of times but no luck.
Thanks for your help and time.

P

QUOTE (p01 @ Apr 19 2005, 07:45 AM)

Hi
hi! how about checking your ligase. The ygive problem quite often. Try changing the enzyme and buffer as ATP in buffer goes bad after repeated freeze thaw. Hope that helps.
prachi
I am trying to clone in an insert of approx 700bp into pCs2+ vector using ClaI. However, I have been completely unsuccescful. I believe the ClaI cuts because when i do a double digestion with ClaI and XhoI I get bands of the right size. However after restricting the vector with ClaI and ligating it I dont get any colonies. Both the vector and the insert have been grown in SCS110 strain. I have also tried diffrent molar ratios. The ClaI is from fermentas any suggestions about where I should buy it from?
Can somebody help me with this please?
I have done it a number of times but no luck.
Thanks for your help and time.

P

If I am not mistakne, ClaI is dam dcm senzitive., you shoul check your bacterial strain.

QUOTE (prachi @ Apr 19 2005, 09:05 AM)

QUOTE (p01 @ Apr 19 2005, 07:45 AM)

Hi
hi! how about checking your ligase. The ygive problem quite often. Try changing the enzyme and buffer as ATP in buffer goes bad after repeated freeze thaw. Hope that helps.
prachi
I am trying to clone in an insert of approx 700bp into pCs2+ vector using ClaI. However, I have been completely unsuccescful. I believe the ClaI cuts because when i do a double digestion with ClaI and XhoI I get bands of the right size. However after restricting the vector with ClaI and ligating it I dont get any colonies. Both the vector and the insert have been grown in SCS110 strain. I have also tried diffrent molar ratios. The ClaI is from fermentas any suggestions about where I should buy it from?
Can somebody help me with this please?
I have done it a number of times but no luck.
Thanks for your help and time.

P

As Blasko says ... ClaI is very sensitive to methylation so it may not be you ligation but your digestion.

Try an alternative Dam- bacterial strain.

Hi,

I know very well the problem of Cla I and it´s sensitivity for Dam- Methylation. The SCS110 cells should work, as they are dam-.
In my opinion, it might be a problem of the transformation efficiency of these cells. It´s 1000-fold lower than that of XL-Blue cells of stratagene.

I think, the cells are not "competent" enough to do direkt transformation.

If both, insert and vector are cut correctly, ligate and transform into more efficient bacs, and, in case you are successful, retransform into the SCS 110, if you need to do further restriction with ClaI.

I did so and it worked.

I hope I got you right....it´s worth to try!


Cheers
claude

Thanks everybody but the sad thing is I tried everything and still

I only used SCS110 to cut out the insert and restrict the vector but for ligation transformation I used DH5a cells.

Am trying again this time with a higher vector to insert ratio...Cant use any other Res sites as we want to myc-tag the insert at the C-terminus.
Incase this helps. I got very few clonies with CLaI cut but no dephosphorylated vector. Cud be a Ligation problem.