RF-Cloning.org Forum

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