202-pET28b-DNase1

pCMVK-StrepII (193)

Make  pCMVK-StrepII

Date: 2011-09-02

Notebook:

Page:

Clone ID: 193

Experimenters: Paul

Annealing

Date:
Anneal 8685-T and 8685-B at 95_⁰C for 2 minutes.
Digest the vector pCMVK-GFP(#194) with BgIII+ Sal1

Run gel and cut out vector,  purify using spin column

Ligate with annealed oligos for 2 hours

Primer-F 8685-T tc gac CACGTG GAATTC TCTAGA TGG AGC CAC CCG CAG TTC GAA AAG TAA CTC GAG GGATCC A
Primer-R 8685-B GATCTGGATCCCTCGAGTTACTTTTCGAACTGCGGGTGGCTCCATCTAGAGAATTCcacgtgg

and transform Top10

Lane 1 shows the digestion of vector (pCMVK-GFP) with BgIII+ Sal1;

Lane 2: the plasmid F2 from # 7896 Kinase, digested with Sal1 + HindIII

Lane 3: the plasmid F02 from # 7898 Kinase, digested with Sal1 + HindIII

Lane 4:digestion of #348

Lane 5: PlentiGP-app, run DNA Argrose gel after miniprep

Colony Screening by Restriction Digestion

1, 2,: Digest with BglII + KpnI

3, 4, 5: 194 digest with BglII + Kpn1

6~10: 193 digestion with BglII + Kpn1

11, 12, 13: 194 digest with Kpn1+ Xho1

14~18: 193 digest with Kpn1 + Xho1

193: digest with Kpn1+ EcoR1

digest with Kpn1 + Sal1

Looks like clone #4 is good.

--Paul.xu 15:44, 4 September 2011 (EST)