Hi, I've cloned my GOI into TA cloning vector( pDrive) vector, after I did transformation into DH5 alpha I got good number of colonies.After this when I'm doing plasmid isolation using conventional protocol, I don't see any band when I run my isolated plasmid sample over agarose gel, but every time I do see something stuck into the well. Please suggest me what might be the reason behind this problem. thanks
Do you have any evidence that your GOI is in the vector? Did you do PCR to check if your cloning worked before plasmid isolation?
Hi Avinash,
I'd would like to know the purpose of running an agarose gel of the plasmid.
How long is yous GOI? What was the strength of the agarose gel you prepared?
Did you include a DNA ladder? Did it separate nicely? If yes, there is no need of troubleshooting the way you prepare the gel. Instead troubleshoot the plasmid extraction protocol. Whatever you see in the well could be impurities.
I would suggest you to first check the GOI in the sample by PCR. After that check the size of plasmid on Gel. if its ok no need to worry. just check the protocol for plasmid isolation or the components of it. particularly lysis buffer.
Ying, Yes I expect my GOI is in plasmid because pDrive vector comes in a linear form and it will not circularize until and unless GOI with poly-A overhang gets integrated with plasmid's poly-U overhang. I'm sure thus transformation was successful because my cells are growing well on antibiotic plates. I did colony PCR of few colonies but I didn't get any product may be because as it is said that there are only 50% chances of integration of GOI with correct orientation. So I'll be trying PCR with more colonies later.
Jacques, I wanted to isolate plasmid so if in future I can do RE digestion so I ran over the agarose gel in order to do gel purification and extraction of plasmid. my plasmid is 3851bp in size and GOI is 946bp. Initially I made gel of 0.7% later I made 1% gel in both cases my plasmid product was stuck in the wells. Yes I included Ladder and it was showing good separation.
I isolated plasmid by normal phenol chloroform extraction method.
Hi Avinash,
I think the answer to the problem you’re facing lies in the way you carried the cloning and plasmid extraction protocols.
Did you do the following while cloning?
1) Amplify your GOI using DNA polymerase that does not possess proofreading activity, such as Taq DNA polymerase. Such polymerases attach a single A overhang to their reaction products, which is crucial for a successful ligation reaction;
2) Add IPTG (50 μM) and X-gal (80 μg/ml) in the agar plates prior to spreading the transformed competent cells. These chemicals are important for blue/white screening of recombinant colonies. Check the attached link for more explanation on it.
Lastly, which reference did you use for the plasmid extraction protocol?
http://www.sigmaaldrich.com/technical-documents/articles/biology/blue-white-screening.html
Jacques, Yes I used DNA Taq without proof reading activity.
I did not use X-gal because I chose antibiotic as selection marker.
Avinash, its always advisable to go through the blue-white screening step. It increasing the stringency of selecting the right orientation of the product. Also, PCR and restriction digest mapping can help you determine whether your GOI is actually where you wanted it to be and in the right orientation. I think Jacques has made a good point!
- Badges
- Science topic