I am trying to clone 3 genes of size 1.041kb ,438 bp,387 bp into a plasmid of 4.7kb size.I have my genes cloned in another expression vector.I did restriction digestion for 30min at 37 C with NEB EcoRI -Hf and BamH1-Hf to cut the genes from its original vector and purified the digested product using TAKARA gel purification kit.The purified products concentration are 20.5 ng/ul ,17.5 ng/ul, 17 ng/ul. I did restriction digestion of my vector for overnight at 37 C with the same restriction enzyme and confirm the linearization with gel shift of the vector .I purified the digested vector and eluted product concentration is 36.5 ng/ul..I did overnight ligation at 16c with t4 dna ligase of thermo Fisher with supplemented ATP of 1:10 molar ratio and I kept vector concentration 20ng. Next day I transformed the entire 10ul ligated product in competent cells.My transformation set up were competence cells without any plasmid, high copy empty plasmid(20ng), digested vector only(20ng) , digested vector plus ligase enzyme, digested vector plus insert .I observed many colonies in high copy number plasmid and no colonies in the other set up in digested vector, and my insert plus vector plate .I got single colonies in digested vector plus enzyme plate.I don't know where I am going wrong?
Earlier I did try with keeping 50ng vector concentration and with t4 dna ligase enzyme from invitrogen at various temperature like 4c overnight 16 C overnight..each time I used to get enormous colonies and afyer doing colony pcr I used get only negative results...
Kindly suggest what should I do and where I am going wrong
Dear Ishmat,
Cloning three genes, one next to another? mcs-Gene1-gene2-gene-3-mcs? If these genes were previously inside an expression host, I assume they may have been expressing three different proteins individually. So, it means they individually would possess a start and a stop codon each. If I have understood your description well, I seriously doubt if you would achieve protein expression. Secondly, the REs, which you have used to excise the genes, are the same for all the three !! So, I doubt the orientation of these, when ligated. Thirdly, may I know what was the reason for you to ligate three genes into a single vector, one behind the other (If I go by your description)? If you aspire to express three proteins (co-expression), I would suggest you to use commercially available plasmids for expression - biCEP - bi-cistronic expression plasmid, is an example. Your approaches of R-D and ligation seem to be okay. But, please clarify your purpose.
Three genes I have to clone in three plasmid not three genes in the same plasmid. And these genes I am taking from another 3 plasmid .
I will like to suggest to use vector: insert at ratio of 1:3 in ligation and decrease amount of your ligation product (like upto 5 ul) in transformation experiments. Also please check carefully to use the right antibiotics in optimum conc. good luck
Thank you sir for your suggestions..I will try this once and see.
I tried with 1:3 ligation ratio and transformed 5ul..the antibiotic is Kanamycin with concentration 50ug/ml I used..but I didn't get any colonies ..........
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