I've been cloning in a 2.4 kb PCR product cut with KpnI/MluI into a ~10kb vector which has a 2.4 kb gene cut out from it with KpnI/MluI. The vector has been gel purified from the fragment. The only difference between the 2 fragments are that the PCR product has been mutagenized so that it now contains an XbaI site in it, which has been confirmed by restriction enzyme digest. The plates look promising, with no colonies on the vector alone plate, 50 colonies on the vector plus ligase plate, and ~500 colonies on the vector plus fragment plus ligase plate. I've screened many colonies, and digested them. I get the digestion pattern of the wild type, which yields a large vector fragment, a 3.7 kb fragment, and a 1.7 kb fragment. What I also get is the large vector fragment, the 1.7 kb fragment and a 1.3 kb fragment. The only explanation that I can come up with is that the vector backbone religated with itself. The new plasmid can be cut with KpnI, MluI, and with both, and yields the same size fragment as the wild type vector digested with KpnI/MluI, except the wild type also has a 2.4 kb fragment, which is the wild type gene. What I don't understand is how the 2 sites are still intact, and how the vector religated. I've been working on cloning this mutated PCR fragment for 3 months with no success. Any suggestions?
-orca-
OK let me get that right...
You are cutting your PCR product = new insert with Kpn and Mlu. You also cut your vector with these enzymes. Then you ligate and digest again with Kpn and Mlu to see if you got the insert or the original. And your question is why all your new insert clones still have the Kpn or Mlu sites?
I think you need to take a day or maybe a week off, relax, have a nice time and mainly sleep. You've definitly been overworking.
If you haven't figured it out by tomorrow, I'll post the answer ok.
QUOTE (orca @ Sep 7 2005, 10:36 PM)
I've been cloning in a 2.4 kb PCR product cut with KpnI/MluI into a ~10kb vector which has a 2.4 kb gene cut out from it with KpnI/MluI. The vector has been gel purified from the fragment. The only difference between the 2 fragments are that the PCR product has been mutagenized so that it now contains an XbaI site in it, which has been confirmed by restriction enzyme digest. The plates look promising, with no colonies on the vector alone plate, 50 colonies on the vector plus ligase plate, and ~500 colonies on the vector plus fragment plus ligase plate. I've screened many colonies, and digested them. I get the digestion pattern of the wild type, which yields a large vector fragment, a 3.7 kb fragment, and a 1.7 kb fragment. What I also get is the large vector fragment, the 1.7 kb fragment and a 1.3 kb fragment. The only explanation that I can come up with is that the vector backbone religated with itself. The new plasmid can be cut with KpnI, MluI, and with both, and yields the same size fragment as the wild type vector digested with KpnI/MluI, except the wild type also has a 2.4 kb fragment, which is the wild type gene. What I don't understand is how the 2 sites are still intact, and how the vector religated. I've been working on cloning this mutated PCR fragment for 3 months with no success. Any suggestions?
-LeserattePD-
QUOTE (LeserattePD @ Sep 8 2005, 05:45 AM)
OK let me get that right...
You are cutting your PCR product = new insert with Kpn and Mlu. You also cut your vector with these enzymes. Then you ligate and digest again with Kpn and Mlu to see if you got the insert or the original. And your question is why all your new insert clones still have the Kpn or Mlu sites?
I think you need to take a day or maybe a week off, relax, have a nice time and mainly sleep. You've definitly been overworking.
If you haven't figured it out by tomorrow, I'll post the answer ok.
QUOTE (orca @ Sep 7 2005, 10:36 PM)
I've been cloning in a 2.4 kb PCR product cut with KpnI/MluI into a ~10kb vector which has a 2.4 kb gene cut out from it with KpnI/MluI. The vector has been gel purified from the fragment. The only difference between the 2 fragments are that the PCR product has been mutagenized so that it now contains an XbaI site in it, which has been confirmed by restriction enzyme digest. The plates look promising, with no colonies on the vector alone plate, 50 colonies on the vector plus ligase plate, and ~500 colonies on the vector plus fragment plus ligase plate. I've screened many colonies, and digested them. I get the digestion pattern of the wild type, which yields a large vector fragment, a 3.7 kb fragment, and a 1.7 kb fragment. What I also get is the large vector fragment, the 1.7 kb fragment and a 1.3 kb fragment. The only explanation that I can come up with is that the vector backbone religated with itself. The new plasmid can be cut with KpnI, MluI, and with both, and yields the same size fragment as the wild type vector digested with KpnI/MluI, except the wild type also has a 2.4 kb fragment, which is the wild type gene. What I don't understand is how the 2 sites are still intact, and how the vector religated. I've been working on cloning this mutated PCR fragment for 3 months with no success. Any suggestions?
-orca-
QUOTE (orca @ Sep 8 2005, 03:02 PM)
QUOTE (LeserattePD @ Sep 8 2005, 05:45 AM)
OK let me get that right...
You are cutting your PCR product = new insert with Kpn and Mlu. You also cut your vector with these enzymes. Then you ligate and digest again with Kpn and Mlu to see if you got the insert or the original. And your question is why all your new insert clones still have the Kpn or Mlu sites?
I think you need to take a day or maybe a week off, relax, have a nice time and mainly sleep. You've definitly been overworking.
If you haven't figured it out by tomorrow, I'll post the answer ok.
QUOTE (orca @ Sep 7 2005, 10:36 PM)
I've been cloning in a 2.4 kb PCR product cut with KpnI/MluI into a ~10kb vector which has a 2.4 kb gene cut out from it with KpnI/MluI. The vector has been gel purified from the fragment. The only difference between the 2 fragments are that the PCR product has been mutagenized so that it now contains an XbaI site in it, which has been confirmed by restriction enzyme digest. The plates look promising, with no colonies on the vector alone plate, 50 colonies on the vector plus ligase plate, and ~500 colonies on the vector plus fragment plus ligase plate. I've screened many colonies, and digested them. I get the digestion pattern of the wild type, which yields a large vector fragment, a 3.7 kb fragment, and a 1.7 kb fragment. What I also get is the large vector fragment, the 1.7 kb fragment and a 1.3 kb fragment. The only explanation that I can come up with is that the vector backbone religated with itself. The new plasmid can be cut with KpnI, MluI, and with both, and yields the same size fragment as the wild type vector digested with KpnI/MluI, except the wild type also has a 2.4 kb fragment, which is the wild type gene. What I don't understand is how the 2 sites are still intact, and how the vector religated. I've been working on cloning this mutated PCR fragment for 3 months with no success. Any suggestions?
-orca-
No, you didn't get it right. I am cloning my fragment into the MluI, KpnI sites. That's true. But then I'm screening with XbaI, KpnI, which should give a 2 kb fragment, and a 1.7 kb doublet, along with vector backbone if the fragment has the mutation. If the fragment is wild type, it should give a 3.7 kb fragment and a 1.7 kb fragment along with vector backbone. However, a lot of my colonies have a 1.7 kb fragment and a 1.3 kb fragment along with vector backbone. My only explanation is somehow the gene is not there and the vector religated without the fragment. My question is, how can the vector religate with incompatible ends (KpnI and MluI) and how it is that I can cut it with both enzymes (assuming it's religated). Thanks for the response, but I don't think it's that simple. I would appreciate any suggestions. BTW, I thought I got it before I went on vacation, and found out it wasn't true when I came back. But I had a nice vacation anyway.
-orca-
QUOTE (orca @ Sep 8 2005, 03:14 PM)
No, you didn't get it right. I am cloning my fragment into the MluI, KpnI sites. That's true. But then I'm screening with XbaI, KpnI, which should give a 2 kb fragment, and a 1.7 kb doublet, along with vector backbone if the fragment has the mutation. If the fragment is wild type, it should give a 3.7 kb fragment and a 1.7 kb fragment along with vector backbone. However, a lot of my colonies have a 1.7 kb fragment and a 1.3 kb fragment along with vector backbone. My only explanation is somehow the gene is not there and the vector religated without the fragment. My question is, how can the vector religate with incompatible ends (KpnI and MluI) and how it is that I can cut it with both enzymes (assuming it's religated). Thanks for the response, but I don't think it's that simple. I would appreciate any suggestions. BTW, I thought I got it before I went on vacation, and found out it wasn't true when I came back. But I had a nice vacation anyway.
Maybe your vector is ligating with itself to form a "dimer". If you run some uncut you should be able to see this. I have had this happen. The only thing I could suggest is to lower the amount of plasmid you use in your ligations or screen lots of colonies!
-ajames-
QUOTE (ajames @ Sep 8 2005, 07:18 PM)
QUOTE (orca @ Sep 8 2005, 03:14 PM)
No, you didn't get it right. I am cloning my fragment into the MluI, KpnI sites. That's true. But then I'm screening with XbaI, KpnI, which should give a 2 kb fragment, and a 1.7 kb doublet, along with vector backbone if the fragment has the mutation. If the fragment is wild type, it should give a 3.7 kb fragment and a 1.7 kb fragment along with vector backbone. However, a lot of my colonies have a 1.7 kb fragment and a 1.3 kb fragment along with vector backbone. My only explanation is somehow the gene is not there and the vector religated without the fragment. My question is, how can the vector religate with incompatible ends (KpnI and MluI) and how it is that I can cut it with both enzymes (assuming it's religated). Thanks for the response, but I don't think it's that simple. I would appreciate any suggestions. BTW, I thought I got it before I went on vacation, and found out it wasn't true when I came back. But I had a nice vacation anyway.
Maybe your vector is ligating with itself to form a "dimer". If you run some uncut you should be able to see this. I have had this happen. The only thing I could suggest is to lower the amount of plasmid you use in your ligations or screen lots of colonies!
I thought about a dimer. The only problem with that is that the new vector is smaller than the old. If it truly was a dimer, it should be larger than the old vector. I cut with only one enzyme and it linearizes to form a fragment that's smaller than the vector with fragment in it. I still don't understand how I get this smaller vector without the gene in it. However, on a happy note, I PCR'd part of the gene and cut it with XbaI so the site seems to be mutated. I think the site is preferentially methylated, so when I digest it, the mutant looks exactly like wild type. The next step is sequencing to make sure this clone is the right thing. Thanks for the response.
-orca-
I am sure you have already thought of this one, but are you getting some sort of partial cutting event with the enzyme when you are screening your clones? maybe the smaller band is supercoiled uncut plasmid? have you run uncut plasmid in a parallel lane? the whole, double-digest buffer issue is maybe at fault?
-aimikins-
QUOTE (aimikins @ Sep 13 2005, 05:33 PM)
I am sure you have already thought of this one, but are you getting some sort of partial cutting event with the enzyme when you are screening your clones? maybe the smaller band is supercoiled uncut plasmid? have you run uncut plasmid in a parallel lane? the whole, double-digest buffer issue is maybe at fault?
I don't think the problem is with partial cutting, because I am actually cutting out a fragment from the vector, and if there was partial cutting, I wouldn't be getting fragment of the right size from cutting. I still don't know how to explain why the vector without the fragment seems to religate, but I have finally gotten my mutant clone (sequenced it). It turns out that all the mutants looked like wild type so I had actually had it all along. The only problem was the Xba I site that has been engineered into the mutation could not be cut due to a dam methylation site close by. When I PCR'd part of the gene and cut it with XbaI, it digested fine. Then, just to make sure, we sequenced it. Bingo! We have learned a lot from this particular cloning experiment. I was pleasantly surprised. Thanks for all your replies. At least the important part of the mystery is solved.
-orca-
