problem with ligation - ligation (Oct/13/2005 )
i am trying to ligate 2kb PCR product in pGEMt vector, i tried several times but i cant, what could be the probable reason? i really appreciate ur suggestions, Thanks a lot
Couple of questions
On the 2kb PCR product -
1. Did you gel purify to assure you are working with the 2kb band and no other foreign PCR species?
2. What restriction sites do you have on each end? How many bp's do you have beyond the Restriction site on each end? When you digest, do you do a multiple digest or one enzyme at a time? How long and at what temp. are you digesting?
3. Have you ran any of your digested insert on a gel?
pGEMt vector questions -
1. How far apart are your restriction sites on the vector?
2. Do you treat with alkaline phosphatase? I assume your using two unique RE's but if you were using only 1...this could be impt.
3. Have you ran any digested plasmid on a gel?
Ligation questions -
1. What ratio are you using insert : plasmid?
2. What temp. and how long are you ligating?
3. Are you running ligated product on a gel and looking for Plasmid + Insert, or transforming and mini-prepping?
i am trying to ligate 2kb PCR product in pGEMt vector, i tried several times but i cant, what could be the probable reason? i really appreciate ur suggestions, Thanks a lot
Couple of questions
On the 2kb PCR product -
1. Did you gel purify to assure you are working with the 2kb band and no other foreign PCR species?
2. What restriction sites do you have on each end? How many bp's do you have beyond the Restriction site on each end? When you digest, do you do a multiple digest or one enzyme at a time? How long and at what temp. are you digesting?
3. Have you ran any of your digested insert on a gel?
pGEMt vector questions -
1. How far apart are your restriction sites on the vector?
2. Do you treat with alkaline phosphatase? I assume your using two unique RE's but if you were using only 1...this could be impt.
3. Have you ran any digested plasmid on a gel?
Ligation questions -
1. What ratio are you using insert : plasmid?
2. What temp. and how long are you ligating?
3. Are you running ligated product on a gel and looking for Plasmid + Insert, or transforming and mini-prepping?
Thanks a lot for ur quick reply! Thanks again. Its really helpful for to think other aspects. I m new in Mol biol field and dont have much practical experience as well.
I had used TBE for initial expt but used TBE modifier while extracting DNA from gel.
1.The ratio of insert:Plasmid i tried 2:1- 66ng insert, 3:1 ie 100ng insert, 4:1 in 2:1
2.for ligation i had kept at 4 overnight and once almost for a day.
3.i didnt check with gel after ligation but did transformation directly
4.Transformation-
i got very few hardly 2-3 blue colonies, 3:1 gave more white colonies,
5. Colony PCR
i did colony PCR, and used primers designed by me to chech the ligation the expected PCR product is 412bp, using amplitaq-gold i felt probably its difficult to get 2kb PCR product using M13, T7 primers so instead i used my own primers that i had designed earlier to check the expected gene. But the PCR product is too faint, so i am suspicious about ligation.
6.Plasmid extraction- alkaline lysis method
so i isolated plasmid 260:280 ratio about 1.608 to 1.688 and cut with ECoRI, i dont know whats the problem, whether the plasmid is supercoiled or what, i couldnt cut it, except in 1 or 2 samples and moreover the electrophoresis showed band around 10kb, and in few 5 kb which is expected vector with ligation of 2kb .
so i tried to cut with Not1, i got 10 kb band and 5 kb band and only one sample, three diff bands of 10kb, 5kb and 3kb, but none of 2kb.
shall i try with multiple RE?
I would like to know , if there r pyrimidine dimers present in the PCR product , how can i reduce/ eliminate them? just by reducing exposure time ... would it be a problem in ligation?
thanking u again.
Using a 3:1 ratio of insert to vector should be good. However I think you may want to increase your ligation temperature. You may want to try 16C O/N or RT for @3hrs. However the rest of your answers I think you have missed my point. Usually when ligation is not working, you need to get back to the basics.
Let me tell you how I would approach the problem from the beginning.
1. Design of primers - Did I incorporate enough bp's beyond the restriction site, so that the enzyme will cut on the end of the oligo?
2. Digestion on oligo - Does either enzyme have star activity? Can they cut in the same buffer? Time of digestion? Now I am assuming that you are using EcoRI and NotI
http://www.neb.com/nebecomm/products/productR0101.asp
http://www.neb.com/nebecomm/products/productR0189.asp
Please read the general notes for each if you are not familiar with them already.
--After digesting the oligo, if you are unsure, run a little bit of it on a gel and check to see that you still have your 2kb band, and not a smear.
3. Digestion of plasmid - Which plasmid are you using?
http://www.promega.com/tbs/tm042/tm042.pdf...9;pGEM%20t'
(pGEM t) or (pGEM t-Easy) verify you are working with the correct one containing the NotI and EcoRI sites. If you are using pGEM t-Easy the NotI and EcoRI sites are sufficiently spaced from each other so that you should see good cutting. Digest the plasmid with the same considerations you put into digesting the 2Kb insert. Run a small sample on a gel to see what it looks like after digestion.
Ligation - I'd do it for 16C overnight, also be sure to run a control with vector only, and transform into your favorite competent cells.
I am confused, is the pGEMt vector a TA cloning vector? I thought it was. Did you purchase it from Promega as a kit? If so, there is no need to digest the vector...you just add your cleaned-up PCR product which you must amplify using taq (this adds the A overhang that allows it to ligate to the T overhang in the pGEMt vector). Also, no need to dephosphorylation..the vector cannot ligate to itself.
i am trying to ligate 2kb PCR product in pGEMt vector, i tried several times but i cant, what could be the probable reason? i really appreciate ur suggestions, Thanks a lot
Couple of questions
On the 2kb PCR product -
1. Did you gel purify to assure you are working with the 2kb band and no other foreign PCR species?
2. What restriction sites do you have on each end? How many bp's do you have beyond the Restriction site on each end? When you digest, do you do a multiple digest or one enzyme at a time? How long and at what temp. are you digesting?
3. Have you ran any of your digested insert on a gel?
pGEMt vector questions -
1. How far apart are your restriction sites on the vector?
2. Do you treat with alkaline phosphatase? I assume your using two unique RE's but if you were using only 1...this could be impt.
3. Have you ran any digested plasmid on a gel?
Ligation questions -
1. What ratio are you using insert : plasmid?
2. What temp. and how long are you ligating?
3. Are you running ligated product on a gel and looking for Plasmid + Insert, or transforming and mini-prepping?
Thanks a lot for ur quick reply! Thanks again. Its really helpful for to think other aspects. I m new in Mol biol field and dont have much practical experience as well.
I had used TBE for initial expt but used TBE modifier while extracting DNA from gel.
1.The ratio of insert:Plasmid i tried 2:1- 66ng insert, 3:1 ie 100ng insert, 4:1 in 2:1
2.for ligation i had kept at 4 overnight and once almost for a day.
3.i didnt check with gel after ligation but did transformation directly
4.Transformation-
i got very few hardly 2-3 blue colonies, 3:1 gave more white colonies,
5. Colony PCR
i did colony PCR, and used primers designed by me to chech the ligation the expected PCR product is 412bp, using amplitaq-gold i felt probably its difficult to get 2kb PCR product using M13, T7 primers so instead i used my own primers that i had designed earlier to check the expected gene. But the PCR product is too faint, so i am suspicious about ligation.
6.Plasmid extraction- alkaline lysis method
so i isolated plasmid 260:280 ratio about 1.608 to 1.688 and cut with ECoRI, i dont know whats the problem, whether the plasmid is supercoiled or what, i couldnt cut it, except in 1 or 2 samples and moreover the electrophoresis showed band around 10kb, and in few 5 kb which is expected vector with ligation of 2kb .
so i tried to cut with Not1, i got 10 kb band and 5 kb band and only one sample, three diff bands of 10kb, 5kb and 3kb, but none of 2kb.
shall i try with multiple RE?
I would like to know , if there r pyrimidine dimers present in the PCR product , how can i reduce/ eliminate them? just by reducing exposure time ... would it be a problem in ligation?
thanking u again.
[quote name='Shubhada' date='Oct 14 2005, 05:33 PM' post='27843']
[quote post='27826' date='Oct 14 2005, 01:26 PM' name='Shubhada']
[quote post='27823' date='Oct 14 2005, 12:05 PM' name='Rafflez']
[quote post='27822' date='Oct 13 2005, 09:53 PM' name='Shubhada']
i am trying to ligate 2kb PCR product in pGEMt vector, i tried several times but i cant, what could be the probable reason? i really appreciate ur suggestions, Thanks a lot
[/quote]
Couple of questions
On the 2kb PCR product -
1. Did you gel purify to assure you are working with the 2kb band and no other foreign PCR species?
2. What restriction sites do you have on each end? How many bp's do you have beyond the Restriction site on each end? When you digest, do you do a multiple digest or one enzyme at a time? How long and at what temp. are you digesting?
3. Have you ran any of your digested insert on a gel?
pGEMt vector questions -
1. How far apart are your restriction sites on the vector?
2. Do you treat with alkaline phosphatase? I assume your using two unique RE's but if you were using only 1...this could be impt.
3. Have you ran any digested plasmid on a gel?
Ligation questions -
1. What ratio are you using insert : plasmid?
2. What temp. and how long are you ligating?
3. Are you running ligated product on a gel and looking for Plasmid + Insert, or transforming and mini-prepping?
[/quote]
Thanks a lot for ur quick reply! Thanks again. Its really helpful for to think other aspects. I m new in Mol biol field and dont have much practical experience as well.
I had used TBE for initial expt but used TBE modifier while extracting DNA from gel.
1.The ratio of insert:Plasmid i tried 2:1- 66ng insert, 3:1 ie 100ng insert, 4:1 in 2:1
2.for ligation i had kept at 4 overnight and once almost for a day.
3.i didnt check with gel after ligation but did transformation directly
4.Transformation-
i got very few hardly 2-3 blue colonies, 3:1 gave more white colonies,
5. Colony PCR
i did colony PCR, and used primers designed by me to chech the ligation the expected PCR product is 412bp, using amplitaq-gold i felt probably its difficult to get 2kb PCR product using M13, T7 primers so instead i used my own primers that i had designed earlier to check the expected gene. But the PCR product is too faint, so i am suspicious about ligation.
6.Plasmid extraction- alkaline lysis method
so i isolated plasmid 260:280 ratio about 1.608 to 1.688 and cut with ECoRI, i dont know whats the problem, whether the plasmid is supercoiled or what, i couldnt cut it, except in 1 or 2 samples and moreover the electrophoresis showed band around 10kb, and in few 5 kb which is expected vector with ligation of 2kb .
so i tried to cut with Not1, i got 10 kb band and 5 kb band and only one sample, three diff bands of 10kb, 5kb and 3kb, but none of 2kb.
shall i try with multiple RE?
I would like to know , if there r pyrimidine dimers present in the PCR product , how can i reduce/ eliminate them? just by reducing exposure time ... would it be a problem in ligation?
thanking u again.
Thats right, i m using pGEMT easy vector purchased from Promega as a kit, the vector need not have to be digested or dephosphorylate as well.
Earlier we had been able to ligate 1.6 kb product to ligate, but this time i couldnt. why ?