May I know if it is true that gene with any sequence also can be inserted into pGEM-T Easy? Since it has AT-overhang, so will this affect the type of gene inserted?
Hi Yen....Yes you can insert any gene sequence in pGEM-T easy vector but one thing you have to remember....you have to amplify your gene using Taq polymerase as the PCR product will give you the same overhang which will bind with the overhang of the vector...unless you can do one thing...if you are doing PCR with Pfu, then you have to add AT tail later.
I can't understand what do you mean by "type of gene inserted". yes it can be inserted through opposite direction also that you have to ensure by sequencing it. But its not a great problem, you can prepare the full contig of your gene sequence by sequencing it from both end (T7 promoter and SP6 promoter end).
I will suggest you one last thing, if you are going for expression from that gene then try to avoid pGEM-T easy vector because later these overhang can cause problem in expression level...Hope it will help you...Best of Luck!!!!
You can look through this link:
http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Manuals/0/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Protocol.pdf
Hi Yen....Yes you can insert any gene sequence in pGEM-T easy vector but one thing you have to remember....you have to amplify your gene using Taq polymerase as the PCR product will give you the same overhang which will bind with the overhang of the vector...unless you can do one thing...if you are doing PCR with Pfu, then you have to add AT tail later.
I can't understand what do you mean by "type of gene inserted". yes it can be inserted through opposite direction also that you have to ensure by sequencing it. But its not a great problem, you can prepare the full contig of your gene sequence by sequencing it from both end (T7 promoter and SP6 promoter end).
I will suggest you one last thing, if you are going for expression from that gene then try to avoid pGEM-T easy vector because later these overhang can cause problem in expression level...Hope it will help you...Best of Luck!!!!
You can look through this link:
http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Manuals/0/pGEM-T%20and%20pGEM-T%20Easy%20Vector%20Systems%20Protocol.pdf
As an alternative, if you didn't do your PCR with Taq Polymerase there is a method to "adapt" your insert. My mentor at UNC gave me this protocol. You may try it if you find yourself in that situation (the way I did):
Are you wondering if there is a way to tail your blunt-ended PCR fragments that were amplified with a proofreading DNA polymerase like Pfu DNA polymerase? Of course there is, and here is what you do.
1. Use 1–7µl of purified PCR fragment.
Note: The fragment must be purified or the proofreading enzyme will continue to remove the A-overhangs created by the Taq DNA Polymerase.
2. Add 1µl Taq DNA Polymerase 10X Reaction Buffer with MgCl2.
3. Add dATP to a final concentration of 0.2mM.
4. Add 5 units of Taq DNA Polymerase.
5. Add Nuclease-Free Water to a final volume of 10µl.
6. Incubate at 70°C for 15–30 minutes. A program called AT Mod was created in the thermocycler for this purpose.
Give it a shot- Worked amazing for me.
The size of the PCR product has a limitation of cloning into pGEMTeasy vectors. Small fragments get cloned easily than larger ones..especially if the PCR product is too large.
With that being said I have cloned a 2,120 BP insert into pGEM-T-Easy.
My insert is 500bp and I used Taq polymerase for PCR. So this one can be inserted right?
500bp should be much easier. But note that, Taq polymerase has no proof reading ability, so you'll need sequencing to confirm there is no mutations in the cloned fragment. Alternatively, you can use Pfu and then add AT tail as suggested above.
Yes, I just want to insert the gene inside and then send for sequencing.
I suggest to use a Thermostable DNA polymerases without proofreading activity to clone your sequence, in order to generate fragments with 3 ́A-tailed fragments.
Our method is even more simplified (lazy us...): do your PCR with a proof-reading enzyme, then in the last 72oC extension step (after cycling has finished) add 1 uL Taq to same reaction. Works like a teat. BTW, the precise biochemical event is that Taq adds a single template-independent 3' terminal A on each of the two strands of the PCR amplicon. The terminal T comes from the pre-cut vector. It can be generated by adding a template-independent addition of T's to blunt ends by TdT or by clever construction of restriction sites. AT tailing does not actually take place.
Good luck,
A.
Hi Teng Tai, 500bp should be easier for insertion. For efficient addition of an A overhang on amplified PCR product, add final extension step for at least 10 min. Then purify this PCR product and proceed with manufacture's instruction for insertion and transformation. This step works for me. Good Luck!
May I know how many genes can pGEM-T carry? I just read through a paper where they insert a promoter and another three genes in it. Is this plasmid stable?
The site of insert and Long insert DNA is effect insertion. PGem T easy suitable for insert Pcr product because it add poly a tail in pcr product. Bigger peace of pcr product hard to insert and ligase affect insertion. Please clean your pcr product before insert.
When do I want to use pGEM T easy only for qPCR quantification? Would they have some methodology to suggest?
With that being said you can modify PCR fragments not amplified by Taq polymerase by a protocol on the Promega webpage.
Hi Debaprasad Parai
What do you mean by " if you are going for expression from that gene then try to avoid pGEM-T easy vector because later these overhang can cause problem in expression level." - what is the reason?
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