Yijia LI 3 5 2018

Date:3/5/2018

3021:1-4 restriction enzymes:BssHII+XbaI

3027:5-8 restrcition enzymes:NotI+NheI

Buffer:Fastdigest

Time:16min

Result:seems good, but the result of sequencing is not,the product were actually are undigested vectors,3016pCEL0,3017pCEH0. so we collected them for other using.

Date:3/6/2018

Subclone of 3027:

Vector:3017pCEH0

Insert:3027pMiniT2

restriction enzymes:MheI+NotI

Time:30 min

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.

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

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.

Subclone digestion of vector 3016pCEL0

Restriction enzymes:BssHII+NheI

Time:30min

Buffer:Fastdigest

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

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.

Date:3/7/2018

Miniprep digestion:

3021pMiniT2:1-4

Restriction enzymes:EcoRI

3161:5-8

Restriction enzymes:NdeI+XhoI

Results: we sent 3021pminiT2 to do sequencing

3161 is not good.

PCR of 947

Lane1,2:Fragment 1 Primer:945 F+947 R

Lane3,4:Fragment 2 Primer:947 F+945 R

Buffer:Platinum HiFi supermix

Results:good, cut out and do next PCR

second PCR of 947

Primer: 945 F+945 R

Buffer:Patinum HiFi supermix

Results:good, cut out the first band 

Date:3/8/2018

Miniprep digestion of 3021:

Buffer:Fastdigest

Time:15Min

Restriction enzymes: BssHII+NheI

Results:the second lane seems like a digested band

Subclone of 3010;vector digestion

Vector:3130

Buffer: Fastdigest

Time:15min

Result:good cut out and combine with insert

Date:3/9/2018

subclone of 3132:

BsrGI+NheI

Buffer:Fastdigest

Time:15 min at 37oC

Result: good, cut out

subclone of 3168:

Restriction enzymes:BsrGI+NheI

Buffer:Fastdigest

Time:15 min at 37oC

Results: good,cut out and combine

subclone of 3152:

Restriction enzymes:BsrGI+NheI

Buffer:Fastdigest

Time:15 min at 37oC

Result: Vector is good, insert is not good, just cut out vector

subclone of 3167:

Restriction enzymes:BsrGI+NheI

Buffer:Fastdigest

Time:15 min at 37oC

Result: Vector is good, insert is not good, just cut out vector

subclone of 3131:

Restriction enzymes:BssHII+XhoI

Buffer:Fastdigest

Time:15 min at 37oC

Results: good,cut out and combine

subclone of 3009:

Restriction enzymes:BsrGi+KpnI

Buffer:Fastdigest

Time:1 hour at 37oC

Result:good cut out bands and combine

subclone of 3135:

Restriction enzymes:BsrGi+KpnI

Buffer:Fastdigest

Time:1 hour at 37oC

Result:good cut out bands and combine

subclone of 3076:

Restriction enzymes:BsrGi+KpnI

Buffer:Fastdigest

Time:1 hour at 37oC

Result:vector is good cut out band, insert is not good.