Editing Yijia LI 3 5 2018 (section)

== Date:3/6/2018 ==

'''Subclone of 3027:'''

Vector:3017pCEH0

Insert:3027pMiniT2

restriction enzymes:MheI+NotI

Time:30 min

[[File:Subclone of 3027.JPG|RTENOTITLE]]

'''Result''':So we cut out the band and did subclone

'''Mini prep Digestion of 3162_3163_3164'''

3162:5-8 Not I+BamH I

3163:9-12 BsrG I+Not I

3164:13-16 BssH II+Nhe I

Buffer:Fastdigest

Time:30 Mins

'''Results:we selected 6,9,16 to do sequencing, all of the sequencing results are good and we labled the plasmid and did bacterial stock.'''

[[File:Miniprepdigest of 3162 3163 3164.JPG|RTENOTITLE]]

'''subclone test:'''

'''because miniprep digestion of 3162_3164_3165 has some good results, so we selected other sites to cut the DNA to doublecheck'''

3162:BssHII+KpnI 330

3163:NotI+KpnI 1101

3164:NheI+KpnI 377

[[File:Subclone test of 3162 3163 3164.JPG|RTENOTITLE]]

'''Test for 3017pCEH0 digestion:'''

'''because after digesting, three bands showed up in the gel.'''

Buffer:Fastfigest

Time:30min

Vector:3017pCEH0

'''Results:NheI may have another sites in the vector. becase NheI cutting has tha same result as NoyI+NheI cutting.'''

[[File:Vector3017 digest.JPG|RTENOTITLE]]

'''Subclone digestion of vector 3016pCEL0'''

Restriction enzymes:BssHII+NheI

Time:30min

Buffer:Fastdigest

Result: not good, did not see the small band.

[[File:Subclone digest of 3016pCEL0.JPG|RTENOTITLE]]

'''945 PCR'''

2X PCR Buffer:Platinum Supermix HiFi

Template:1281

Primer:945 F+ 945 R

'''Results:good, cut out and did gel purfication to prepare next step.'''

[[File:945PCR.JPG|RTENOTITLE]]