Hello, everyone. I'm trying to subclone a 5 kb gene to a vector (approx. 5 kb). Initially, I designed the primers which have restriction enzymes overhang (XhoI and XbaI). I amplified my gene using those primers (I took 50 ng of the template) and run agarose gel to check the band. Unfortunately, I got smeared band but the size is fine. So I decided to gel purify. Followed by double digest with XhoI and XbaI. Again, I gel purified to eliminate other stuff.
On the other hand, I digested my vector (6 ug) with XhoI and XbaI for 2 h and CIP for 45 min. Followed by gel purification. Finally, I ligated my insert to this vector.
I used DH5a for transformation. Unfortunately, I did not get colonies.
I was wondering what could be the reason for no colonies. Also, I wanted to know how to clone this insert.
Please shed some light on this cloning. Many thanks in advance.
Arun.
Hi Arun
What vector you are using? is that capable of taking 5kb insert? good Transformation protocol is required for high 5KB gene is very important. Did you get colonies in control plates(Using only vector)? that will tell your transformation has occurred successfully and need to concentrate on ligation protocol. Since you got smear band during amplification it is better to redo it again to confirm your gene amplification.
Buy a TOPO TA cloning kit. They are a bit pricey, but they work every time, even for novice users. Don't bother with all of those digests, CIP, cleanups, etc. Use a DNA polymerase that leaves an Adenine overhang on your PCR product and you can directly ligate it into the plasmid.
Thank you for your suggestions.
@Preethy: I'm using pCS4 plasmid. I did not try self-ligation. I will try next time.
@Sebastian:
Primer details: I designed FP and RP to amplify my gene of interest and added the restriction enzyme sequences flanking to the primers. Also, I added 5 bases at the ends. So the total of 25 bases in a primer.
For PCR amplification: I took 20 or 50 ng of the template to amplify my gene and got the smeared band. I used proof-read enzyme (pfu) to do the job. I kept 5 min for elongation.
Ligation: I took 7 ul of the purified insert (I did not calculate the conc) and 200 ng of the vector to ligate overnight at room temperature. I used T4 DNA ligase from NEB.
Transformation: I used dh5a. After heat shock, I left it on ice for 5 min and 1 h for regeneration at 37 deg.
@Katie: Thank you for your suggestion. If nothing works well, then I will go for TA kit.
For efficient ligation go for overnight ligation and followed by gel extraction of ligation mix.
To do the above you need to have good concentration of vector and insert as well as the ligation reaction mix volume should be minimum of 100UL.
Use different ratios of vector and insert and as well as try the Quick Ligase enzyme for efficient ligation.
You need to clone the gene into the pGEMT Easy vector using TA cloning kit. After verifying by sequencing, cut the gene, purify it from the gel and re-clone it into your target vector.
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