I'm trying to clone a ~500 bp gene into the pET28a plasmid. I first cloned this gene into the pCMV6-AC-DDK plasmid (using AscI and XhoI) to incorporate the DDK tag. This was successful verified by sequencing.
I am following the same basic procedure for amplifying the gene + DDK using primers that add NcoI (N-term) and NotI (C-term) sites but I am not having any luck. I have double checked my primers and they appear to match up with the regions I am interested in, and the Tm's appear to be ok. I purify after PCR and then do a double digest for 4h (at least 6 bases are included so the restriction site isn't at the very end). After the digestion I gel purify and I see a nice band around 500 bp. The pET28a was digested similarly and again yields a nice band on a gel.
To ligate (T7) I have been using an excess of the gene insert (approximately 4:1). I have tried 25 C for about 20 minutes followed by overnight at 4C. I have also tried ligating using a custom PCR routine that is closer to the Tm of the sticky end regions (10C 1 min, 14C 1 min, 25 C 1 min; x60, followed by a 10C hold). I transformed 4 uL of both of these ligations into 50 uL of XL10 Gold cells (fresh) and plated the entire transformation reaction. I used the uncut pET28a as a control and saw many colonies with it.
I am fairly confident that I have amplified the correct PCR product because I see a solid band on the gel. Should I try to change the ratio of insert to plasmid in the ligation? I am transforming a larger amount of the ligation (4uL) which might effect the efficiency of the transformation but would it completely inhibit it?
Any suggestions on where to trouble shoot or what the most probable cause of failure might be?
my overhang and restriction site sequences that I am adding with the primers are as follows:
Nterm NcoI - GACGAC CCATGG
Cterm NotI - CGGGCCGC TAATATGC
Hi Michael,
You should increase the time of your double digestion of your PCR product to at least 20 hours+ because Not I needs that amount of time to achieve 90% cleavage of the ends. With only 4 hours of digestion you are not creating enough Not I ends for the ligation to occur on that side.
Additionally, transforming 4ul of the ligation is a lot but so long as you are not trying to transform more than ~40ng of DNA (which can inhibit transformation) you should be fine in that respect.
Good Luck!
I have experienced similar problem.
In my case, I solved the problem using "infusion" instead of restriction enzyme.
Just amplify the insert with infusion primers that you design and cut the band from agarose gel.
Then mix the insert and enzyme cut empty vector with infusion premix.
Infusion reaction takes only 15 mins then transform.
If you can't obtain the colony, there is a possibility that the vector is not digested well.
Then you can amplify the empty vector with inverse PCR and use it for infusion reaction.
Off course you have to buy infusion kit and design the primer for infusion reaction but I believe it helps you.
http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17497
Sorry, Rob. That was a typo. The primer does indeed have GCGGCCGC. Good catch though.
I will first try a longer digest as Tom suggested.
Update:
I did an overnight digestion (37C) followed by an overnight ligation (4C) which gave colonies. Sequencing confirmed cloning was as planned. Thanks for your help.
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