Using pGEMT Vectors - (Oct/09/2008 )
I have never used pGEMT vectors for ligation before (or carried out a ligation) and I am little confused on the protocol on using them.
What is the control insert DNA? Am I supposed to change the volumes of one of the components for optimisation?
Thanks in advance!
What is the control insert DNA? Am I supposed to change the volumes of one of the components for optimisation?
Thanks in advance!
I think it is the control included in the pack. You can just ignore it.
Previously, I just set up my ligation as they have suggested and just add in 3uL of purified PCR product without any dilution. Total reaction volume is 10uL.
I use 250 bp PCR product amplified using Taq polymearse as my positive insert.
But you need not to use it because when your colonies were grown over LB agar you can pick it and do
colony PCR with Sp6 and T7 primers.
P.S.
Do not forget to use Taq because if you use only Proof-Reading enzyme the PCR product will not have 3'-A overhang.
Hi Kara,
It is fair to say that if you only have a single PCR product, you can directly use a few uL of your PCR mix for the ligation. However, if you are unsure of the specificity of your PCR reaction, do a gel extraction and clone the eluted DNA into pGEM-T.
As its name suggests, pGEM-t Easy should be quite straightforward, and it is if you follow these guidelines :
- measure the concentration of the DNA bit your want to clone, ideally, dilute it to 20-25 ng/uL if it is under 1kb
- use a molar ratio of insert-to-vector of 3:1, that is 25ng of an insert of 500bp, as suggested on page 13 of the pGEM manual. All you have to optimize is the amoun of DNA insert you add to the reaction setup, it will depend on the size and concentration of your DNA as explained in the pGEM manual, which you SHOULD have read before posintg here
- the control DNA in the pGEM kit is intended to make you buy more kits from Promega, because you are using reagents for nothing, it only comes to be useful to make ligation controls with/without insert DNA and with/without ligase when you do serious cloning
- use a control plasmid for the transformation step if you an unsure of the competency of your cells.
don't forget to put ampicillin and xgal in your agar...
It turns out we don't need to use it and so we were not told about it (it wasn't even in the kit!). I only asked as I read the manual and got confused as to why we were not told. Should be easy now. Thanks for your help!