Help, no insert, ligation product couldn't be cut anymore! - (Dec/26/2008 )
Hi all,
I am having some problems with my cloning...I RE (Nde1 and Xho1) an insert from pET-28c plasmid and ligate it with pET-21a vector (double digested with the same two restrition enzymes). Every step looked perfet: both orignal vevtors can be double digested nicely (gel shows clearly), DNA purification is succefully (the insert and vector show pure on Gel), after ligation by using Takara DNA ligation kit (30min @ 16 degree C), about the 30 colonies showed up after transformation. I selected all of them and did plasimid isolation. Just to check if the insert is there, I double digested the isolated plasmid, BUT, not only there is NO INSERT, but also none of those plasmid could be double digested any more 
!!!! Every plaimid still shows three band after double digestion! The enzymes still works because I did the control and they can cut my original vector nicely! What's can be wrong?
I used the CIP from sigma (added 1ul and incubate 1 hr at 37 degree C). Nde1 and Xho1 are from invitrogen (using REact buffer2). Since none of them has star actity, I did overnight.
Any tips?
Thanks a lot in advance!
Several things could have gone wrong.
1- the DNA extraction was poorly conducted. As a result the DNA was so dirty it could not be cut.
2- the digest is incomplete. Are you certain this is uncut plasmid and not an incomplete digest.
3- given the low recovery rate (30 colonies), these colonies are likely to be products of vector molecules that have recircularised in some strange manner, resulting in the lost of the NdeI and XhoI sites. Thus the NdeI and XhoI enzymes are not able to cut the plasmid.
Thus when testing for DNA fragment insertion, use one enzyme that cuts within the vector (not the restriction site use in the ligation) and one enzyme that cuts the insert (not the restriction site used in the ligation.)
CIP has be handled with care. Overdephosphorylation with CIP will lead to damage to the ends of your vector. Given what you have quoted, I feel that the vector has been over dephosphorylated. Given that the ends your cut vector are incompatible I would suggest avoiding CIP. It is not an absolute requirement in your ligation strategy. CIP can be more trouble than it is worth.
I would suggest redoing the ligation using vector (NdeI/XhoI) cut vector that has not been dephosphorylated.
The normal recovery rate for a successful ligation is 100s of colonies.
I have thus far not see a ligation with the conditions of 30min at 16 Celsius. Does it work?
I has seen 30min at 25C. 5hr at 16 C, or overnight at 16C. I would raise the temperature to room temperature if you are ligating for only 30min. Or leave the ligation mix to ligate for several hours at 16 C.
1- the DNA extraction was poorly conducted. As a result the DNA was so dirty it could not be cut.
2- the digest is incomplete. Are you certain this is uncut plasmid and not an incomplete digest.
3- given the low recovery rate (30 colonies), these colonies are likely to be products of vector molecules that have recircularised in some strange manner, resulting in the lost of the NdeI and XhoI sites. Thus the NdeI and XhoI enzymes are not able to cut the plasmid.
Thus when testing for DNA fragment insertion, use one enzyme that cuts within the vector (not the restriction site use in the ligation) and one enzyme that cuts the insert (not the restriction site used in the ligation.)
CIP has be handled with care. Overdephosphorylation with CIP will lead to damage to the ends of your vector. Given what you have quoted, I feel that the vector has been over dephosphorylated. Given that the ends your cut vector are incompatible I would suggest avoiding CIP. It is not an absolute requirement in your ligation strategy. CIP can be more trouble than it is worth.
I would suggest redoing the ligation using vector (NdeI/XhoI) cut vector that has not been dephosphorylated.
The normal recovery rate for a successful ligation is 100s of colonies.
I have thus far not see a ligation with the conditions of 30min at 16 Celsius. Does it work?
I has seen 30min at 25C. 5hr at 16 C, or overnight at 16C. I would raise the temperature to room temperature if you are ligating for only 30min. Or leave the ligation mix to ligate for several hours at 16 C.
I deeply appreciate your suggestion!
Plasmid isolation is unlikely to be wrong since I have done this too many times, and never got a singe problem.
I am not sure if the double digest of vector was complete, even though I did it for overnight, but this cannot explain the loss of the cutting site. I can double the enzyme amount.
The overdephosphorylaiton is a good point! I might try to redo it without CIP or at least recuce the reaction time. I thought the use of CIP was always a requirement.
. Ligation at 16 Celsius for 30min is the manual suggestion from that kit.
Again, thank you so much!
Happy new year!
1- the DNA extraction was poorly conducted. As a result the DNA was so dirty it could not be cut.
2- the digest is incomplete. Are you certain this is uncut plasmid and not an incomplete digest.
3- given the low recovery rate (30 colonies), these colonies are likely to be products of vector molecules that have recircularised in some strange manner, resulting in the lost of the NdeI and XhoI sites. Thus the NdeI and XhoI enzymes are not able to cut the plasmid.
Thus when testing for DNA fragment insertion, use one enzyme that cuts within the vector (not the restriction site use in the ligation) and one enzyme that cuts the insert (not the restriction site used in the ligation.)
CIP has be handled with care. Overdephosphorylation with CIP will lead to damage to the ends of your vector. Given what you have quoted, I feel that the vector has been over dephosphorylated. Given that the ends your cut vector are incompatible I would suggest avoiding CIP. It is not an absolute requirement in your ligation strategy. CIP can be more trouble than it is worth.
I would suggest redoing the ligation using vector (NdeI/XhoI) cut vector that has not been dephosphorylated.
The normal recovery rate for a successful ligation is 100s of colonies.
I have thus far not see a ligation with the conditions of 30min at 16 Celsius. Does it work?
I has seen 30min at 25C. 5hr at 16 C, or overnight at 16C. I would raise the temperature to room temperature if you are ligating for only 30min. Or leave the ligation mix to ligate for several hours at 16 C.
I think another way to see if ligation is successful would be to do a PCR to see if your insert is inside.