pGEM T easy vector for cloning - (Sep/20/2006 )
Hi ....
Well i checked the activity of ligase enzyme and buffer by religating the 100bp restricted ladder. please check the first gel picture....got a streak for liagtion of 100bp ladder and a single band for Ecor1 restricted lambda DNA....so the ligase works fine....
then again i did the ligation with the A-tailed PCR product of mine with the vector (check the second gel picture) no ligation.....
there is no ligation with even the positive control insert promega gave with the kit....
the last 4th well is the -ve control with the vector alone.....
so what do i conclude from this??????
Thankyou
Hi
Anybody has any suggestions....still trying to find the problem....out....
Yes, change your strategy. There is no point doing the same thing over and over again. Maybe use a TOPO kit or design primers with restriction sites for sticky end cloning into pUC or pBluescript.
M.
Anybody has any suggestions....still trying to find the problem....out....
I agree with ML1975. Change strategy.
M.
Hi
Anybody has any suggestions....still trying to find the problem....out....
Hi...
Iam new with the TA cloning ....In the protocol for TA cloning the pUC is restricted with EcoRV or Sma1...i suppose iam right????
I have the protocol taken from protocol online of TA cloning....Anyother protocol which will work with a new hands on....
Tell me one thing when we purify the PCR product with promegas SV purifying wizard kit...do we loose the A overhangs???
Trying to concentrate the PCR product that i have so that i can correctly prove that the pGEM T vector is not working fine.....
Thankyou....
Iam new with the TA cloning ....In the protocol for TA cloning the pUC is restricted with EcoRV or Sma1...i suppose iam right????
I have the protocol taken from protocol online of TA cloning....Anyother protocol which will work with a new hands on....
Tell me one thing when we purify the PCR product with promegas SV purifying wizard kit...do we loose the A overhangs???
Trying to concentrate the PCR product that i have so that i can correctly prove that the pGEM T vector is not working fine.....
Thankyou....
if you are amplifying your fragment with taq then it will contain the A-tails. The percentage of adenylated to non-adenylated fragments varies. To blunt-end clone into pUC, you digest your vector with blunt-end cutters smaI or EcoRV. Then dephosphorylate the vector, do some research on the web..there are a number of enzymes like calf intestinal which is hard to inactivate and cumbersome or shrimp alkaline phosphatase or antarctic phosphatase, both of which are heat inactivated. You can dephosphorylate your vector and run it through the ligation without removing the buffer or purification if you use the heat inactivated phosphatases. You could try adding your PCR product to see if the ligation works, or you may need to treat the PCR product with mung-bean nuclease to remove the A-tails. Alternatively, if you amplify your PCR product with a proof-reading polymerase like pfu, then the product will be blunt ended with no A-tails. Finally, you PCR product will have to be kinase treated with ATP so that it contains phosphates at the ends. This is necessary for ligase to cataylse ligation to the vector. This is a start but you should research all this so that you understand how it all works. Catalogues that sell these products are a good source of information.
Hi ...
I did another experimentation with pGEM T easy vector system 1.....
As the promega tech serve said that the PCR product concentration is very low....i made the pcr product concentration higher by precipitating the pcr product and then purifying it again precipitating the product from this purified sample and dissolving this in juz 15uL sample that makes up a real gud concentration .....after this i did the ligation and the gel pictures of a whole 10uL ligation sample was run but dint do transformation its was coz i wanted to get a complete picture of the bands though faint managed for a picture. the resukts i get in gel picture..
1. in first lane it is 1Kb ladder
2. in the second lane is the PCR product+ vector - in that lane there are three bands one of the ligated product around 3.8kb, second of the vector, and the third of the PCR product (801bp-0.8kb).
3 in the third laneis the +ve control provide along with the kit. there are two faint bands one in the region of 3Kb that is of vetcor and one band in 3.5Kb that is of the control insert with vector.
well my question is i have used the same sample before for transformation but havent got transformation no white colonies at all both in the Pcr product and also in +ve control insert...
can someone tell me what is the concentration of the ampicillin u have added in the selection media...
thank you
Hi all...
somebody have any suggestions...please reply...
Thankyou
i am using 100ug/ml
Iam facing a problem with cloning. Iam using the pGEM T easy vector for cloning. Followed the ligation protocol as per promega instuctions. I tried to transform the ligated vector into E.coli DH5 Alpha by electroporation but got no results. Promega has provide with a positive control insert along with the kit. when the electroporated cells of ecoli were plated got no white colonies nor in the +ve control nor in negative control (only with the pGEM vector alone, no blue colonies). Thot the cells were not competent and went for calcium chloride method of transformation using Ecoli JM109 this time we also used pUC18 as a negative control besides the pGEM vector alone. The trasformation with pUC gave blue colonies but no blue colonies in the pGEM vector alone and no white colonies in +ve control promega provided nor in our insert.
Can anyone suggest what would go wrong is it with ligation step?? or is it with transformation?? iam unable to get the right reason where it would go wrong??? guide me.
Did you aliquote the ligation buffer the first time you opened it? If not, that might be the reason. It is quickly becomming unusable if you thaw and defreeze it.
Also, make sure to use a heating block or preferably a water bath, not a heating incubator og PCR-machine for the incubation at 45 degrees during transformation!