[Help] New vector construct problem - (Dec/13/2008 )
Dear All,
Hi, I'm currently working on the new T7 vector(~4k) construct with the interested gene A (>1kb).
Background information: T7 vector is from a T7 plasmid with geneB. Gene A is from the other vector.
I did the PCR by introducing two new enzyme restriction sites (NdeI and HindIII) to the interested gene A, digest the insert with two enzyme for 4hrs and then cut the insert gene out from the gel;
Meanwhile, I cut the T7 vector using the same two new enzymes, and cut the vector out. Also, I purify the gene B out of the gel as well.
Ligase the insert (geneA) and T7 vector with (1:1) and (3:1) ratio at three conditions: 1)4 degree, o/n; 2)16 degree, 4hrs; 3) RT, 1hr. Then take 5ul ligation into 75ul competent cell for further transformation.
I could get colonies. But interestingly, I ended up with the cloning with T7 vector + geneB after sequencing and enzyme digestion test with NdeI and HindIII. Not T7 vector with geneA as expected.
In order to make sure there is no contaminant of gene B and uncut T7 plasmid. I repeated again, and I got the very similar results...
I am now very confused. It looks to be a very traditional method, but I could not think of which part it wrong. I would like to ask you to help me to give some comments, please?
Also, for the PCR primers, I could not think anything wrong with them. Since I also ran the PCR of gene A on gel, it is at the right level, which is also different from gene B.
I wish to get some progress before Christmas.
Thanks in advance.
Hi, I'm currently working on the new T7 vector(~4k) construct with the interested gene A (>1kb).
Background information: T7 vector is from a T7 plasmid with geneB. Gene A is from the other vector.
I did the PCR by introducing two new enzyme restriction sites (NdeI and HindIII) to the interested gene A, digest the insert with two enzyme for 4hrs and then cut the insert gene out from the gel;
Meanwhile, I cut the T7 vector using the same two new enzymes, and cut the vector out. Also, I purify the gene B out of the gel as well.
Ligase the insert (geneA) and T7 vector with (1:1) and (3:1) ratio at three conditions: 1)4 degree, o/n; 2)16 degree, 4hrs; 3) RT, 1hr. Then take 5ul ligation into 75ul competent cell for further transformation.
I could get colonies. But interestingly, I ended up with the cloning with T7 vector + geneB after sequencing and enzyme digestion test with NdeI and HindIII. Not T7 vector with geneA as expected.
In order to make sure there is no contaminant of gene B and uncut T7 plasmid. I repeated again, and I got the very similar results...
I am now very confused. It looks to be a very traditional method, but I could not think of which part it wrong. I would like to ask you to help me to give some comments, please?
Also, for the PCR primers, I could not think anything wrong with them. Since I also ran the PCR of gene A on gel, it is at the right level, which is also different from gene B.
I wish to get some progress before Christmas.
Thanks in advance.
Hi,
How big is gene B?
There should be some uncut vector in your ligation which may have lead to such a problem. I would suggest you to try doing the digestion of the vector with controls. You can use digestions with single enzymes parallely to make sure that the enzyme is working.
The gene B has the similar length as Gene A but smaller.
Uncut vector could be the reason to lead this problem, I will try to avoid this. And I will take your advice to do single cut to make sure. Thanks.
In addition, I just realised today that I didn't acturally clean up the PCR product before digestion. I guess that my PCR product didn't get digested well, so it leads to the failure of further ligation...
Hmmm, what do you think?
I'm going to repeat this tomorrow.
Uncut vector could be the reason to lead this problem, I will try to avoid this. And I will take your advice to do single cut to make sure. Thanks.
In addition, I just realised today that I didn't acturally clean up the PCR product before digestion. I guess that my PCR product didn't get digested well, so it leads to the failure of further ligation...
Hmmm, what do you think?
I'm going to repeat this tomorrow.
good luck!
Uncut vector could be the reason to lead this problem, I will try to avoid this. And I will take your advice to do single cut to make sure. Thanks.
In addition, I just realised today that I didn't acturally clean up the PCR product before digestion. I guess that my PCR product didn't get digested well, so it leads to the failure of further ligation...
Hmmm, what do you think?
I'm going to repeat this tomorrow.
yes, I've been told that you should always purify on gel the PCR product before digestion.
I purify again after digestion ( no gel, but directly on the column ), maybe I'm doing too much purification,m but at least it works.
good luck