3161 pET28b-GST-Myc-HA-Flag-V5

Date: 3/4/2018

Performed by: Paul

subcloning

miniprep digested with Nde1-Xho1

Subcloning: Nde1+ Xho1

Vector: 1803 pCold-6His-GST-(E)-(3C)-PKARIIb (insert is 1329bp)

insert: 3159, 225 bp with Nde1 + Xho1

3161:5-8

Restriction enzymes:NdeI+XhoI

Results: we sent 3021pminiT2 to do sequencing

3161 is not good.

Miniprep of 3168_3132_3161

3168:1-4 BsrGI+NheI

3132:5-8 BsrGI+NheI

3161:9-12 NdeI+XhoI

Buffer:Fastdigest

Time:30Min

Result:#7 seems good, so sent to do sequencing, the rest of them are suspicious

Date:3/14/2018

Miniprep digestion of 3131_3161_3167

Name length Enzymen Buffer

3131:1-4 342 BssHII+XhoI Cutsmart

3161:5-8 225 NdeI+XhoI Cutsmart

3167:9-12 397 BsrGI+NheI NEB 2

Time:40min

Miniprep digestion_3161_3168

3161:1,2 NdeI+XhoI Buffer:Cutsmart

3168:3 BsrGI+NheI

Buffer:NEB 2

Time:40min

Result:seems good, do sequencing

Redo miniprep of 3131_3161_3167

3131:1-4 BssHII+XhoI 342 Cutsmart

3161:5-8 NdeI+XhoI 225 Cutsmart

3167:9-12 BsrGI+NheI 397 NEB 2

Time:40min

Restriction Digestion Analysis of 3159 pMiniT2-1FRG-HA-VL-kappa

by Paul, 3/16/2018

Nde1 + Xho1 digestion: 1803 (vetor)  and 3159 (insert)

Left: vector 1803

Right: 3159, there are too many bands, too complicated

Cut out EcoR1 band, middle ~ 1kb, purified with mini silica column, elute with 20 ul H2O

digest with Nde1 + Xho1

3/17/2018, cut out the smallest fragment, notice 1803 vector is overdigested, can not be used

digest vector 1803 with Nde1-Xho1 (right lane)

Date:3/19/2018

Miniprep of 3161

Restriction enzymes:NdeI+XhoI

Buffer:Fastdigest

TIme:30min

Result:combine the rest of them except#2 to do sequence

Date:3/20/2018

PCR screening of 3161

Lane1,2:length 1300bp

Primers:

820-His-HA-His-T;

T7-term(R)

Lane 3: length 294

Primers:pCold-seq-F;

T7-term(R)