What are your optimization strategies when cloning with a single enzyme at both ends?
What if you have 2 inserts?
Did you ever try to join the insert and a PCR product using chimeric internal primer (0.01mcM) with 15bp overlapping to one and 15 to the other piece, plus 5 and 3' primers (2mcM)?
Would you rather synthesize the whole insert as 1 molecule instead?
Hi Antonio,
I think a type IIs restriction enzyme like BsaI might be of use to you if I understand your question correctly. Because the recognition site is different to the cleavage site you can pretty much make any sticky ends you like.
I must confess though that these days I now just buy pretty much all my DNA synthesised and expression optimised. When you factor in your time in addition to reagents it is also possibly cheaper.
Best of luck
Andy
You approach the problem by 3 ways
1. chemical synthesize your genes as like primers and use it for further cloning
2. Design overlapping primers and amplify first round of amplification with these primers then purify longest amplicon and reamplify it with end primers or vice versa.
3. amplify the two inserts separately purify them, dephosphorylated one of them purify it and use it for ligation with other fragment if the restriction site is present in junction of two sequences . Then after ligation use the ligated product for pcr confiramtion with end primers if confirmed go for cloning.
I don't know whether I understand your question correctly, but when you want to create multimers using only one restriction site in your plasmid you can use the combination BamH1/Bgl2 or Sal1/Xho1. These respective pairs have the same sticky end, but different 6bp recognition site, ie 1st and 6th base is different. So a BamH1-Bgl2 fragment can be cloned into a BamH1 site and only one end will recreate a functional BamH1 site, which can be used to clone another BamH1-Bgl2 fragment, etc. Same goes for Sal1-Xho1 fragments. These are the only pairs for this approach I know.
Thank you for your answers, since I am creating a vector with 4 genes (separated by 2A seq), I inserted gene #3 using MluI at both ends, and plus I created a Asc1 site in 3'. Therefore, the 4th gene will be inserted by ligating Asc1 at both ends.
It is a similar strategy as using neo-schizomers enzyme, as most of you suggested. That is a good point for future refs. I spot those enzyme-pairs=
• BamH1/Bgl2
• Sal1/XhoI
• AatII/ ZraI
--However, for my cloning, b/c a 2-inserts ligation maybe difficult, I am ligating the oligo and PCR first=
1. oligo (Mlui-Afe1) (from overlapping N)...http://gcat.davidson.edu/igem10/
2. a PCR product (afe1-Mlui).
My inserts size is about 2K kb.Hope this works, if not I will try Infusion.
-Lastly, to join the oligo and the PCR i tried also a chimeric-PCR-approach according with those reference papers=
A. Methods in mol biol, 67, Genevieve Poont-Kingdon;
B. Nucleic acid research 17, 12, 1989, Jeff Von).
PS: maybe before trying the In-fusion from Clontech, the next question could be: is better using a synthesized DNA or G-block as compared with an annealed oligo. I would not think so.
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