to directly answer your question: the ONLY difference between the pGEMT and pGEMT-easy is the multiple cloning site. they BOTH have 3' T overhang for TA cloning (not blunt end!!).
https://www.promega.ca/products/pcr/pcr-cloning/pgem-t-easy-vector-systems/?catNum=A1360
*there are protocols to blunt the plasmid (for blunt insert) or alternatively, you can add an A overhang to your blunt insert
As with all cloning experiments, doing controls will help you understand whether your experiment is working as expected. Doing ligations with vector alone and of vector plus control insert (I think the pGEM-T kit provides template and primers for a control) are a good start. For TA cloning, one problem can be that after long term storage or multiple freeze-thaw cycles, the plasmid can lose the terminal T overhangs. When this happens, the frequency of recircularized vector increases - and this can be seen as an increased number of colonies in a self-ligated vector control. This background can mean you need to screen (many) more colonies to get ones with insert.
As far as the size of plasmids is concerned, unless you are digesting the plasmid DNA, I don't think you can gain much from trying to estimate plasmid size from agel of supercoiled DNA. There are several factors that cause problems. First, supercoiled DNA doesn't run predictably alongside linear markers (although you can buy supercoiled markers); second, nicks and cuts in supercoiled DNA can give rise to open circular (non-supercoiled circles) and linear plasmid molecules that tend to run more slowly than supercoil; third, many of the high copy-number vectors (like pGEM) can exist as multimers in the cell, which can produce bands of double, triple the size of the original vector. Thus having a number of bands in one lane doesn't indicate muliple plasmids - the bands may just be different forms of the same plasmid.
For normal TA (and other) clonings my group routinely use chemically competent JM109 from Promega (as supplied with the pGEM-T cloning kit). For more difficult cloninings - including very large inserts I sometimes use electrocompetent DH5alpha.
The only difference is an extra 15bp of multiple cloning region giving a wider choice of restriction enzymes see :http://www.promega.com/resources/protocols/technical-manuals/0/pgem-t-and-pgem-t-easy-vector-systems-protocol/
as you want to cloning vectors, you can not miss the following paper, for efficent work.
Zhang, Gang and Tandon , Anurag. Quantitative Models for Efficient Cloning of Different Vectors with Various Clone sites. Available from Nature Precedings (2012)
Gang Zhang University of Toronto
There are a difference of 15 pb but with more restriction enzyme targets in the pollinker which sometimes is very convenient.
Hi Francisca. Can you please give me the name of the enzyme that you use to amplify you PCR product? I need amplify a PCR product with 2400 bp. Thanks!
@K.S. Sim any should do - 8 kb isn´t that much.... pGemT for instance...
A general comment about cloning long PCR products - first, always use an enzyme with good processivity and low error rate. Taq polymerase alone wont be good enough and you will struggle to get clones with the correct sequence. I've use Roche Expand Long Template and KOD HotStart in the past for PCR products up to 10kb or so with good results. Second, for cloning longer (PCR) fragments it may also help to use a positive selection system like the Life Technologies zero-background. My experience has been that this can increase the chances of getting back difficult (large) clones. Also, if you want to use blunt ended PCR products in a TA cloning system, you can easily add A overhangs by treating the (pure) PCR product with Taq polymerase. Just incubate in PCR buffer with enzyme and dNTPS. No denaturation, just 72C for 15 minutes will be enough to do the job.
@Gabor,
The cloning efficiency not very good.
I'm doubt that will 10k plasmid supercoils into 3k or else ? I extracted alot plasmid, more than 94% of the plasmid at 2.5kbp size. haiz..
Just hv one with 4 bands, one at 10kbp, 7kbps, 6kbps, and brightest one at 4kbps.
@George,
thx alot. Should i use Electro-poration or TSS or CaCl ??
there rumor said promega pGEM-T is not efficient as the past.
As with all cloning experiments, doing controls will help you understand whether your experiment is working as expected. Doing ligations with vector alone and of vector plus control insert (I think the pGEM-T kit provides template and primers for a control) are a good start. For TA cloning, one problem can be that after long term storage or multiple freeze-thaw cycles, the plasmid can lose the terminal T overhangs. When this happens, the frequency of recircularized vector increases - and this can be seen as an increased number of colonies in a self-ligated vector control. This background can mean you need to screen (many) more colonies to get ones with insert.
As far as the size of plasmids is concerned, unless you are digesting the plasmid DNA, I don't think you can gain much from trying to estimate plasmid size from agel of supercoiled DNA. There are several factors that cause problems. First, supercoiled DNA doesn't run predictably alongside linear markers (although you can buy supercoiled markers); second, nicks and cuts in supercoiled DNA can give rise to open circular (non-supercoiled circles) and linear plasmid molecules that tend to run more slowly than supercoil; third, many of the high copy-number vectors (like pGEM) can exist as multimers in the cell, which can produce bands of double, triple the size of the original vector. Thus having a number of bands in one lane doesn't indicate muliple plasmids - the bands may just be different forms of the same plasmid.
For normal TA (and other) clonings my group routinely use chemically competent JM109 from Promega (as supplied with the pGEM-T cloning kit). For more difficult cloninings - including very large inserts I sometimes use electrocompetent DH5alpha.
pGEM-T is for cloning of blunt PCR products obtained by Pfu polymerases and thair derivatives
PGEM-T-Easy is for cloning of PCR products by Taq polymerase.
@george,
I did the insert control given by pGEMT kit and ~4kbp circularize pGEM-T as control as well. The cloning efficiency is ok. The kit didnt come with primer. The pGEM-T vector is new.
It gonna waste alot of enzyme if screen every clone.
I face one more problem that my digested plasmid band is not identical to my digested PCR (gel purify) fragment. >_
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