Ligation problem with PCR product cloning - (Jan/22/2008 )
I did PCR to get a 2kb gene of interest and i used Taq pol and got a very strong band of 2kb. however the gene of interest has SalI and PstI restriction sites. later I did digestion for the insert and the vector PQE-9(34kb) with SalI and PstI and used buffer3+BSA accourding to NEB, overnight digestion.
next day, I did ligation with T4 ligase(insert:vector= 1:1 and 3:1) for overnight.
finally I transfomed it to TGI cells using electroporation and plate the cells.
nextday, i did colony PCR and got 2kb, ~35kb, and >9kb. so I plate the same single cell that I used for colony PCR and did double digestion with SalI and PstI. but I got the 35kb only. I dont know what was the problem. i did so many times colony pcr for the other cells and cant get the 2kb. what i think is that the vector religated to itselfe or to a small fragment.
the primers that were used in colony pcr were for a specific sites in PQE-9 vector, where the two primers surround SalI and PstI.
can anyone suggest what to do?
kalthoom
Can you give us a bit more info about your protocol? Did you SAP treat your vector?
actually i did not do SAP treatment to my vector.
and here is the protocol:
first of all i did PCR purification for the PCR samples. then i checked on nanodrop (34.6 ng/ul) and run on 0.8% agarose gel, and i got only one band which is about 2kb. then i did digestion with PstI and SalI for the insert and the vector: i used NEB restriction enzymes (PstI and SalI ), 100x NEB BSAand 10xbuffer3- i did negative control for only vector. i kept the samples in 37C incubator O/N. next day, i did PCR purification and ligation with T4 ligase and used 10x ligation buffer for O/N in room temp. but before adding T4 ligase i heated the samples using pcr ( 90C 2min, 60C 3min, 20C 4min, 20C 7min).
the day after , i did PCR purification and transformation using electroporation. i used TGI cells. here is the protocol for transfrmation:
first transfer 2ul from the ligation samples to a cold cuvette and then add 38ul of TGI cells and mixed gently and electroporate----250uF, 1.7 volts (time const~4.6).
immediately added pre-warmed SOC media (1ml).
incubate at 37C with shaking for 1 hr.
spin down cells-resuspend in 50ul. then plate at 50ul on LB-AMP plates.
incubate in 37C incubator O/N.
next day i picked colonies and resuspend in a 50ul H2O {1.5ml microcentrifuge tubes}.
NOTE: i left each colony in milli Q water for about 15 min and then mixed gently.
i used some for colony pcr, then run on 0.7% gel and got these bands:
2Kb
~35Kb
>9Kb
i used taq polymerase. i got these bands in all the colonies except the band which is > 9Kb was in one of the colony.
the primers i used were for PQE9 vector not the primers that recognise the insert. maybe i should had use the insert primers (these primers has a rest site for SalI and PstI).
so i plate the cells. I also inoculate colonies and incubate 37C shaking. did miniprep (used qiagen) and then restriction digestion witth PstI and SalI.
these are the bands i got after digestion:
~35kb for all of the colonies.
does this mean my insert didnt ligated with the vector?
what i think that the 2Kb band i got in all colonies (COLONY PCR) where for the TGI cell genome. i also think that the vector ligated to itself. would it be better if i did SAP treat first, but isnt for blunt end?
hope this detail would be helpful.
cheers
I apologize for the wrong info about the sizes of PQE9 vector and the band i mintioned.
corrections:
PQE9 vector size is 3.4kb
the band that was visualized on gel after colony pcr is ~3.5kb.
cheers
You need some controls to figure out what is happening. Some things I would do:
* think about switching to an enzyme other than SalI if possible
* Avoid overnight digestion of your DNA. 1 hour or even 1/2 hour should work and give less trouble
* Heat kill your enzymes after digestion (see the spec sheet, typically 80C for 20 minutes)
* Your colony PCR sounds as if it is being done with far too many cells. It should be impossible to get a 35Kb fragment with a sane extension time, so you are probably seeing genomic DNA. Touch a 10 ul tip to a colony and resuspend in 50 ul. Add 1 ul of this to a PCR reaction and plate 2 ul onto a master plate. Less is more in this case.
* Think about the relative amounts of vector and insert. They should be roughly equimolar in the final ligation.
* You should be able to simply mix these DNA fragments, the T4 buffer, and very small amounts of T4 ligase and keep them at room temperatue for 1/2 hour and then transform.
* Check that the T4 ligase buffer is still good with a control
* Run a tranformation control using serial dilutions of the plasmid you are going into. You should have efficiencies of 10^9 or more colonies/ug of plasmid with electroporation.
* You should be able to do this in a day, not three days, with more likelihood of success
* think about switching to an enzyme other than SalI if possible
* Avoid overnight digestion of your DNA. 1 hour or even 1/2 hour should work and give less trouble
* Heat kill your enzymes after digestion (see the spec sheet, typically 80C for 20 minutes)
* Your colony PCR sounds as if it is being done with far too many cells. It should be impossible to get a 35Kb fragment with a sane extension time, so you are probably seeing genomic DNA. Touch a 10 ul tip to a colony and resuspend in 50 ul. Add 1 ul of this to a PCR reaction and plate 2 ul onto a master plate. Less is more in this case.
* Think about the relative amounts of vector and insert. They should be roughly equimolar in the final ligation.
* You should be able to simply mix these DNA fragments, the T4 buffer, and very small amounts of T4 ligase and keep them at room temperatue for 1/2 hour and then transform.
* Check that the T4 ligase buffer is still good with a control
* Run a tranformation control using serial dilutions of the plasmid you are going into. You should have efficiencies of 10^9 or more colonies/ug of plasmid with electroporation.
* You should be able to do this in a day, not three days, with more likelihood of success
hey Phage thanks . i was wondering about some points you mintioned.
about the third point you mintioned "the heat one". do you mean for colony pcr? or ligation?
and for the 4th point actually it is 3.5kb not 35kb, its my mistaken when i mintioned 35kb.well... think it is the vector and the the band which is more than 9 kb is the template for sure.
point 6. because im doing electroporation, it would be easier to transform the gene+vector to TGI cells after purification and that is because of salts. I tried before doing lectroporation direct after ligation and didnt work.
i think its a good idea to do serial dilutions for the plasmids. thanx again
Your template should be invisible on a gel after PCR. If it is not, then you are using WAY too muich template DNA. You only need (in principle) one molecule, although I'd recommend quite a few more. This could be one of the problems.
Something I didn't mention was to check that there are sufficient 5' bases in your primer beyond the restriction enzyme cut site. You should have at least six bases beyond the cut site, more is better.
In point 3 I suggested heat killing of your restriction enzymes. If you are not familiar with the concept, check the NEB catalog or google it.
Instead of purifying the ligation reaction, you can drop dialyze it to remove salts. See the Millipore web site on drop dialysis. I like to heat kill the ligase before transformation, but this is probably unnecessary.
Something I didn't mention was to check that there are sufficient 5' bases in your primer beyond the restriction enzyme cut site. You should have at least six bases beyond the cut site, more is better.
In point 3 I suggested heat killing of your restriction enzymes. If you are not familiar with the concept, check the NEB catalog or google it.
Instead of purifying the ligation reaction, you can drop dialyze it to remove salts. See the Millipore web site on drop dialysis. I like to heat kill the ligase before transformation, but this is probably unnecessary.
Thanks a lot
I have been doing a lot of PCR Cloning and gave up on ligation a long time ago. Too much time and too many problems. Now I use a cloning kit by BPS Bioscience. It uses homologous recombination between your vector and your insert. It allows you to insert your pcr product to your desired vector without going through any intermediate vectors or using ligation. The whole reaction takes about 40 minutes and then you are ready to transform your vector with insert into competent cells. I've found the efficiency to be very high too. Now I only need to screen two colonies per plate compared to 4 in the past when I used ligation.
hope this helps!
I had the problems with salI before. avoid it if you can. I change to KpnI on mine. it worked perfect.
hope this helps!