Editing Yijia LI 3 19 2018

== Date:3/19/2018 ==

'''PCR of 3165 F3'''

Primers:3165 F1+3165 R2

Template:F1+F2

2XPCR Mix:Platinum

Extension time:30s

Annealing:55oC

'''Results:Cut out band and do the next step'''

[[File:IMG 3619.JPG|RTENOTITLE]]

'''Miniprep of 947_947pMiniT2_3131_3152'''

947:1-4 XbaI+NcoI-HF 1394 Cutsmart

947pMiniT2:5-8 ECoRI Cutsmart

3131:9-12 BssHII+XhoI 342 Cutsmart

3152:13-16 BsrGI+KpnI 371 NEB 2

'''Result: sent lane 2, lane 16 to do sequence'''

[[File:IMG 3621.JPG|RTENOTITLE]]

'''Miniprep of 3166pMiniT2'''

Restriction enzyme:SalI+BsrGI

Buffer:NEB 3

Time:30min

[[File:IMG 3620.JPG|RTENOTITLE]]

'''3129 digestion as vector'''

[[File:IMG 3622.JPG|RTENOTITLE]]

'''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]]

'''Redo Subclone'''

'''3131''':BssHII+XhoI

Buffer:Cutsmart

'''Results:cut out the middle band of insert, and cut out the vector.'''

[[File:IMG 3626.JPG|RTENOTITLE]]

== Date:3/20/2018 ==

'''PCR screening of 947'''

Lane 1,2:pT7A3-seq-F;945-R

Lane 3,4:945-F;945-R

Length:1326bp

Extension time:1.5min

2X PCR Mix:Platinum SuperMix

[[File:IMG 3628.JPG|RTENOTITLE]]

'''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]]

'''PCR Screening of 945'''

Lane 1,2: pT7A3-seq-F;945R

Lane 3: 945 F; 947R

extension time:1.5min

2X PCR Mix:Platinum

length:1300 bp

[[File:IMG 3631.JPG|RTENOTITLE]]

== Date:3/21/2018 ==

'''Miniprep of 3166(from PCR)'''

Restriction enzymes:BsrGI+NdeI

Buffer:Fastdigest

Time:30min

'''Result:#1,#3,#5 have bands'''

[[File:IMG 3638.JPG|RTENOTITLE]]

'''Screening of 3166 miniprep'''

1,3,5: BsrGI+SalI

1'',3'',5'':BsrGI''

Time:15min

Buffer:Fastdigest

'''Result:good'''

[[File:IMG 3643.JPG|RTENOTITLE]]

'''Miniprep of 3165pMiniT2_3165_3131'''

3165pMiniT2:1-5 EcoRI

3165:6-10 BsrGI+NdeI

3131:11-15 BssHII+XhoI

Time:30min

'''Result:3165pMiniT2(1-5) have bands,3131(12-15)have bands.redo 3165 digestion.'''

Date:3/21/2018

[[File:IMG 3637.JPG|RTENOTITLE]]

Date:3/21/2018

'''Redo digestion of 3165 from PCR'''

Restriction enzymes:BamHI+BsrGI

Length:151bp

Time:15min

Buffer:Fastsdigest

'''Result:#2,#4 have good bands'''

[[File:IMG 3639.JPG|RTENOTITLE]]

'''Screening of miniprep_3165'''

2,4: BsrGI+SalI

2'',4'':BsrGI

Time:15Min

'''Result:seems good, Smear may caused by high concentration, around 300 ng/ul.'''

[[File:IMG 3642.JPG|RTENOTITLE]]
[[Category:My ELN]]