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

Date: 3/4/2018

Performed by:&nbsp;Paul

subcloning

[[File:3162 3164 3163 3161.JPG|RTENOTITLE]]

miniprep digested with Nde1-Xho1

[[File:3161 3165 947.JPG|RTENOTITLE]]

Subcloning: Nde1+ Xho1

Vector: 1803&nbsp;<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; background-color: rgb(251, 251, 239);">pCold-6His-GST-(E)-(3C)-PKARIIb</span> (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.

[[File:Digestion of 3161 3021pMiniT2.JPG|RTENOTITLE]]

'''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

[[File:Miniprep of 3168 3132 3161.JPG|RTENOTITLE]]

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

[[File:Miniprepof 3131 3167 3161.JPG|RTENOTITLE]]

'''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'''

[[File:Miniprep of 3161 3168.JPG|RTENOTITLE]]

'''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

[[File:Redo miniprep of 3131 3161 3167.JPG|RTENOTITLE]]

== Restriction Digestion Analysis of 3159&nbsp;<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pMiniT2-1FRG-HA-VL-kappa</span> ==

by Paul, 3/16/2018

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

[[File:1803 3159 Nde1 Xho1.JPG|RTENOTITLE]]

Left: vector&nbsp;1803

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

[[File:3159 EcoR1.JPG|RTENOTITLE]]

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

digest&nbsp;with Nde1 + Xho1

[[File:3159 1803 Nde1 Xho1.JPG|RTENOTITLE]]

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

[[File:1803 3159 digestion.JPG|RTENOTITLE]]

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

[[File:IMG 3623.JPG|RTENOTITLE]]

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)

[[File:IMG 3630.JPG|RTENOTITLE]]
[[Category:Cloning]]