Hello,
I have to clone 3 pieces together:
1. 2 Pcr made- the inserts. One 4200bp (13 ng/microliter), one 460bp(21ng/microliter)
2. Vector 3800bp(13ngng/mictoliter).
All pieces were cut with restriction enzymes and run on gel and then purified . The enzymes I used were bpal, mlul, bsu36i.
I tried different ratios and nothing worked. I used Neb quick ligase and also tried overnight with the regular t4 from Neb.
I need help, thank you.
there are some issues and suggestion that i give you..
1. there are no extra sequence on the RE site( like (TTTT)GAATTCNNNN) to optimize the digestion so the product could not be properly digested and cause the ligase cannot ligate the product.
2. what enzyme do you use for PCR?
3. have you purified the product?
4. if youre already success in doing PCR with 4200 bp of product you could just do an overlaping PCR so you will make 1 insert (4660 bp) that you will ligate with the backbone so you will do a regular ligation.
5. you could try golden gate assembly.
Thanks. Ive been using Q5 for the Pcr.
I purified it using the qiagen kit after PCR then digested then purified using monarch NEB kit. By doing this I avoided the gel purification.
What is 3 piece gibson? Thank you very much! Sarina
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning