pGEM T easy vector for cloning - (Sep/20/2006 )
Hi all,
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.
Company cells if threated properly are always very competent.
In the second transformation, using calcium chloried, did the DNA used come from the leftovers from the first transformation reaction?
If you haven't already done so, could you verify that there is DNA actually present in the ligation samples. And that ligation has actually taken place. I have the feeling that the ligation reaction is not working, please check or toss your ligase buffer and T4 ligase. Both reagents go off very easily and rather quickly. The ligase buffer can be checked by its smell. If the smell is faint or absent, then the buffer is about dead.
(only with the pGEM vector alone, no blue colonies)
I am puzzled by this statement, could you elaborate.
(only with the pGEM vector alone, no blue colonies)
I am puzzled by this statement, could you elaborate....
I had used the pGEM vector as a -ve control that is the vector was added along with ligase and buffer.
Is it by running on the gel that we confirm the ligation has taken place???how do i confirm the ligation has taken place from the gel picture which we get???if on an agarose gel what percentage is it the normal 0.7-0.8% we run for confirmation of ligation????
the total ligation reaction was 10ul of which 3ul was used for transformation.
In the second transformation using cacl2 we used the rest 3ul of the ligation reaction. nothing was used from the first transformation.
Tell you te whole process.
We used the pcr product purified using SV gel wizard purifiying kit.
This purified product was used for ligation with pGEM T easy vector. added 3ul of the purified pcr product for ligation reaction.
Added T4 DNA ligase and the ligase buffer provided along with the kit.
In the kit they had provided a control insert as a positive control.
Totally we had the ligation reaction with 1. pcr product 2. control insert DNA 3. -ve control with the vector only.
The first two reaction after transformation shud give white colonies we got none and the -ve control to give blue colonies we got none. thot no transformation using electroporation.
went to follow the classical method of cacl2 using another strain of ecoli JM109 b4 we used ecoli dh5 alpha...thot the strain not proper and changed to JM109 which promega recomends...we brought no competent cells from them...
went for transformation with the same ligation mixture. added 3ul to the cacl2 competent cells of JM109...this time we wanted to c whether it was problem with transformation so we use the pUC vector also as a negative control...
the results were like this no white colonies with 1. pcr product 2. the +ve control provide by promega 3. no blue colonies with -ve control (pGEMT vector) but the transformation had taken place with -ve control of pUC and we got blue colonies. we confirmed that transformation is taking place but..there is some problem.....
Please confirm me how do i test how the ligation reaction has taken place????
kindly reply Thankyou
Lately , we have had ligation problems with pGEM Easy T vector.
First we didnt get any colonies, then we got both white and blue colonies but there was no insert. repeated it and again the same. We couldnt solve the problem yet. Something seems to b completly turned off.
So now we r digesting the PCR product and ligating it directly into a vector. This seems to b working.
Ops.. sorry.
All one needs to do is run the ligation mix on a gel. The gel density is dependent on the insert size and vector size. The idea is to visualise the DNA molecule produced by the ligation reaction. So for a 10 ul ligation mix, run perhaps 5ul on a gel. If ligation is successful, higher MW bands will appear in addition to the already present insert and vector. This will confirm some manner of ligation has occured (though it doesn't say if the ligation between vector and insert has happened.)
First we didnt get any colonies, then we got both white and blue colonies but there was no insert. repeated it and again the same. We couldnt solve the problem yet. Something seems to b completly turned off.
So now we r digesting the PCR product and ligating it directly into a vector. This seems to b working.
Hi,
The ATP present in ligase buffer is very sensitive to temperature. So it may work if you give an additional ATP in your ligation reaction.
Hi....
Iam in real trouble.... 
There is no transformed colonies in the LB amp plates after cloning the insert DNA into pGEM T easy vector system1. The positive control they have supplied (white colonies) and the negative control with only the vector is giving no blue colonies...... 
But when the JM109 is tranformed with PUC iam getting blue colonies????
somebody help ot something is not working.....when the gel is run there is no band shift coming up...the vector(3kb), insert (1.5kb) are seen separately in the gel.....what to do????
Iam in real trouble....
There is no transformed colonies in the LB amp plates after cloning the insert DNA into pGEM T easy vector system1. The positive control they have supplied (white colonies) and the negative control with only the vector is giving no blue colonies......
But when the JM109 is tranformed with PUC iam getting blue colonies????
somebody help ot something is not working.....when the gel is run there is no band shift coming up...the vector(3kb), insert (1.5kb) are seen separately in the gel.....what to do????
Possible problems
-over dephosphorylation has destroyed the end of the vector...
-ligase buffer has gone bad
-ligase has gone bad.
My first suspect would be the ligase and the ligase buffer. Change both components with new stuff and try again.
And a general reminder always aliquote the ligase buffer, and use one aliquote and keep the rest tightly capped and frozen.
pGEMT contains T-overhangs so doesn't require dephosphorylation. What are you using to amplify your insert? Remember that it must be amplified with a taq polymerase to ensure you get A-overhangs. This enables the PCR product to be ligated into the vector. If you are amplifying with a proof-reading polymerase (eg. pfu or PWO) then you can still treat the PCR product with taq to add the A-overhangs. All this is written in the pGEM manual.
Hi....
(My first suspect would be the ligase and the ligase buffer. Change both components with new stuff and try again. )
I have changed both the ligase and ligase buffer with new components...but still iam not getting results...
I use Taq polymerase for amplifying the pcr product and not proof reading enzymes....so there is no problem even with the A-overhangs.....
if it was a problem of transformation...tried both chemical and electroporation both have worked with PUC....and i have blue colonies in the LB amp plates....
tried using the full 10uL ligation reaction containing only with the vector...but dint get blue colonies at all in the ampicillin plates...
i have completely aliquoted the ligation buffer as 5uL into individual tubes when i got the ligase buffer....
where cud i go wrong...
(.....over dephosphorylation has destroyed the end of the vector.....)
can u please explain to me how this cud happenover dephosphorylation???
what more cud go wrong???