Hi everyone!
I am trying to clone a sgRNA into a pX459 but at the end I do not have colonies.
To anneal the oligos I use
sgRNA fwd (100uM) 1uL
sgRNA Rev (100uM) 1uL
10x T4 Ligation Buff 1uL
H2O 7uL Total 10uL
37°C/30min
95°C/5min
Ramp down slowly to 25°C
25°C/5min
And ligation
30ng of BbsI cut gel purified vector
2ul annealed oligos
1uL 10x ligase buffer
1uL t4 DNA ligase
H2O
16°C overnight
And then I transform competent bacteria (XL1 blue)
What could be the problem to not having colonies after transformation?
Thanks in advance :)
Thanks Amy Johnson
for your answer.To transform I use the annealed oligos into 100uL of xl1 blue competent bacteria.
Then, 30 min on ice. After that, 40 min at 42°C and 5 min more on ice. Then I add 200 uL of LB broth (without antibiotic) and 1 hour at 37°C to recover the transfected bacteria. and finally I plate on LB/100ug/uL plates and 37°C overnight.
I actually didn't run a gel after ligation. I will do this the next time!
And which conditions do you use for ligation? 16°C during 1 hour?
I also saw a protocol that uses 22°C during 3 hours, 16°C during 12 hours and 70°C during 10 min.
Thanks!
Hi Diego Benitez Riquelme, have you been able to rule out the problem? I am having the same problem at the moment.
Thanks.
Gio Fidelito I still have the problem.
Now I am trying with this protocol: https://www.versiti.org/Custom/Files/Versiti/8f/8f9e4f84-c20f-4225-a068-45250ee0bd80.pdf and on Wednesday I should have some colonies.
Which protocol do you use?
Hi Diego Benitez Riquelme , I have tried that protocol as well, but I still can't get any colonies. Let me know how it goes for you.
Did you end up finding what works best? Diego Benitez Riquelme Gio Fidelito
Hii Gio Fidelito, have u figured it out what went wrong ? I have been trying to clone gRNAs into px459 vector using F. Zhang lab protocol and in the end I didn't get even a single colony.
Harshit Kalra I actually ended up getting this protocol to work. Make sure your guide overhangs are EXACTLY the same as described in the protocol.
Hii Fahed Abu-Hijleh. Thank you for your reply. I have checked, overhangs are correct. I am doubting that problem is with ligation as I am not using high concentration T4 ligase as suggested by the protocol but the problem is I am not getting colonies in non-insert too. what could be the problem ? Have you got colonies in non-insert ?
Harshit, if you are getting no clonies from your postive control (px459 uncut plasmid), then you are having difficulties with transformation.
- Make sure you are using the recommended competent E. coli cell line. Follow the manufacturers transformation procedure.
- Make sure you are using the correct concentration of ampicilin for your agar plates.
If you are still having problems, perhaps you can sequence the uncut plasmid to make sure what you really have is px459.
You need to use the Stbl 3 E.coli cells for it. Any other cells even Stbl2 will not give you any result (I mean transformation). I have used the same protocol just the bacterial strain is Stbl 3 from Thermo. It always worked.
Thanks,
I have previously performed this protocol, I got no single colony after transformation. I repeated it yesterday, I used 1ug of pX459 and incubate for 1 hour and proceed with the rest of the protocol. I got few colonies but yet to screen for positive insert. The major issue that has not been well define is the duration needed for digestion, and how to avoid under- or over-digestion of vector. Other aspect of the protocol seem straightforward.
Debanjan Mukhopadhyay Sir, can I use the DH5 alpha stain for carrying out transformation using the same pX459 plasmid?