Hi,
I am trying to ligate/insert the phosphorylated/annealed guide sequence into the Bbs1 digested CIP treated px459. I am getting the bacterial colonies on the AMP agar plate. but when I am trying to confirm the Ligation by PCR i am not getting the product. As suggested by my supervisor, I am using F primer (5’-actatcatatgcttaccgtaac-3’) same used in px330 and the R primer same as my bottom guide (starts with 5'-aaac-3'). I doubt the pcr protocol especially the primers I am using. If anyone working px459, please share your thoughts and possibly share the sequence.
Thanks
Naveen
Does the primers anneal to the vector or insert or both? Or you can try using the primers that you used to amplify the insert to confirm the insert
Thanks Wong for your inputs, Primers are designed to anneal with vector (forward) and insert (reverse). we are not getting the product in PCR. could you give your thoughts
Hi Naveen,
I haven't used pX459 myself so far, but for pX330/pX335 I simply do a restriction digestion in stead of PCR analysis to confirm if my gRNA has been insterted correctly. As a successful insertion will destroy the BbsI sites it is possible of performing an BbsI / AgeI double digest to discriminate between positive and negative clones. Clones with insertion will show only linearised plasmid of ~8510bp (only AgeI will be able to cut) Clones without insertion will show a ~980bp and ~7520bp fragment (both BbsI and AgeI will be able to cut) both enzymes work well in NEB buffer 1.
Cheers, Max
Thanks Max, your approach looks more simple. If possible could you please send me the brief protocol you use for this digestion, i mean how many molecules of enzyme you use, incubation time etc.
Naveen
Hi Max, your Idea worked and I got results as you explained. Is was very helpful. Thanks
Naveen
Hi Naveen,
My appologies not answering your previous question in time, but I am happy to hear you figured it out :-) .
Good luck with your CRISPRs!
Max
Hi Max, I need some more help.
I am using electroporation to transfect 661W cells with px459. I am loosing too many cells due to electroporation as i am trying to optimize the protocol. My doubt is "Can I use "Lipofectamine" based transfection for px459/px330 in CRISPR ?" Thanks
Naveen
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