203-pET28bTurboTEV

This is done by Paul

TurboTEV gene synthesis using Kapa HiFi

4/7/2012: One step:

4/8/201: two steps

first reaction:

tube one: oligo 1-10, 0.25 ul (0.5 unit) Kapa

tube two: oligo 11-20, 0.25 ul (0.5 unit) kapa

25 ul final volumn without amplifying primers (outer primers). Amplify for 5 cycles.

while they are still hot (95oc), transfer tube 2 to tube 1, add 2 ul of outer primer mixture (F & R).

Amplify 25 cycles.

4/8/2012

digest PCR product with EcoR1 + Sal1, digest pET28b with EcoR1 + Sal1

cut out/purify bands and ligate

Select 6 clones to mini-prep
203 plasmids are digested by EcoR1 + Sal1

203 plasmids are digested by Xho1

203-1~203-6  may be correct.

Select 203-3 to sequence.