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Hi Steve,
Thanks for the awesome method and even more awesome Website and your help!
I created an account and saved some protocols there but basically, I've been trying to clone PCR products that are around 550-600 bp into pShuttle Vector which already has some other insert inside (Im trying to replace that insert which is 1,6 kb). But I can't seem to get a clone in most of my Transformation attempts. (one time I got clones but they were all negative.)
My 2 step protocol is: 98C 1min, 98C 10 sec, 72C 3min40 sec, 72C 5min (the middle section repeat 18X)
I save an aliquot (10ul) before DpnI Digestion and save another 10 after DpnI Digestion to see if I can see a smear (I see but very faint.) I ran them on gel.
I saw that Long extention time (longer than necessary) is detrimental so I'm gonna stick to 3min 10sec, I will reduce the number of cycles to 15X and normally I transform the entire tube (20 ul) with 50ul of chemically competent dh5alpha.
What else am I missing? Any help is much appreciated
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Hi Asli,
I found three projects attached to your userid, swapping out luciferase for D4, B6, and E3_5.
Your cycling conditions look fine to me, but you are going to have a low efficiency reaction because the plasmid is getting a bit big and the replacement of large fragments usually makes things worse.
Another concern I have is the IRES in between the luciferase and GFP genes. The strong secondary structure can get in the way of proper annealing and extension.
I often encourage people to use In-Fusion, when possible, and I would suggest the same to you. You have a ScaI and NotI pair that will knock out most of the luciferase gene and linearize the parental for the InFusion reaction.
Aside from that, all I can really suggest is poking around more in the forum to see some of the troubleshooting tips people have had through the years. You might have success with some additives.
I wish you the best of luck,
-Steve
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