Yijia Li 3 12 2018
Date:3/12/2018
[edit]3010 miniprep:
Restriction enzymes: BamHI+KpnI
Time:15min
Buffer:Fastdigest
Result:good, sent to do sequencing.#4
Miniprep of 945pMiniT2_947pMiniT2_3131_1727_1834
945pMiniT2:1-4 EcoRI
947pMiniT2:5-8 n EcoRI
3131:9-13 BssHII+XhoI
1727:14 XbaI+NcoI
1834:15 XbaI+NcoI
Buffer:Fastdigest
Time:30Min
Results:945pMiniT2 and 947pMiniT2 are good, so sent them to do sequencing. 3131 is not good, 1727 has no band, 1834 has one band.
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
Subclone of 3168
Retsriction enzymes:BsrGI+NheI
Time:30MIn
Buffer:Fastdigest
Results:good, combine and do the step.
Subclone of 3131:
Restriction enzymnes:BssHII+XhoI
Time:30min
Buffer:Fastdigest
result:good, combine and do ligate
Date:3/13/2018
[edit]Test of 3165miniprep
Digest enzyme:BsrGI
Time:30min
Buffer:fastdigest
Results:not good, got three bands
Repeat subclone of 3152
Restriction enzymes:BsrGI+NheI
Buffer:NEB2
Time:30min
Result:Vector digest is good, but insert is not good.
Test of 3165 miniprep digestion
1:SalI+BsrGI NEB2
2:SalI+KpnI Fastdigest
3:BsrGI NEB2
Time:30min
Digest of Vector 3130
made for Gibson mix
BamHI+KpnI
result:good, cut out and purify
PCR of 947
1281 as template
F1:945F+947R
F2:947F+945R
Extension time:1.5min
Annealling:55oC
Cycles:25
Volumn:25ul
2XPCR MIx:Platinum mix HiFi SUpermix
Results:good,combine F1+F2 and do the next step
Digest of 3009pMiniT
Enzymes:BsrGI+NheI
Time:1hour
Buffer:NEB2
Digest of 30090pMiniT
Enzymes:XhoI+NheI
Time:30min
Buffer:Fastfigest
Date:3/14/2018
[edit]947 F3 PCR
F3:1,2
Negative Control:3
PCR Mix:Platinum
Primers:945F+945R
Result:Have bands, cut the top one and do the next step.
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
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
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
miniprep digestion 3021
3021pMiniT2:BssHII+XbaI 374
3021:BssHII+XbaI 374 Cutsmart
3165:SalI+BsrGI 702 NEB 3
Time:30min
Subclone of 945
1:1727 XbaI+NcoI 173 Cutsmart
2:1834 XbaI+NcoI 173 Cutsmart
3:945pMiniT XbaI+NcoI 1326 Cutsmart
4:3009pMiniT BsrGI+NheI 371 NEB 2
Time:30min
Result:1727 can see band as vector, 945pminiT2 can see band as insert, cut out and combine
Redo Subclone of 3167
Vector:3163pCEH0
Insert:3027pMiniT2
BsrGI+KpnI
Buffer:NEB 2
Time:30min
Result:Cut out and combine
Date:3/15/2018
[edit]Gibson mix practice: 3001
miniprep digestion: BamHI+KpnI
Time:15moin
Buffer:Fastdigest
MiniPrep digestion of 3131
Enzymes:BssHII+XhoI
Buffer:Cutsmart
Time:40min
Result:2,3,4,seems good, do sequencing,select #3
3009pMiniT2 digestion as Insert
Result:suspicious,Redo
Subclone of 947 Vector digstion Time:30min
Buffer:cutsmart
Vecotor:1727
XbaI+NcoI
Result:good, cut out and purify
3009pMiniT2 digestion test 1
Restriction enzymes:BsrG I+NheI
Buffer:NEB 2
Time:30min
Result:seems Lane 4 has band
3009pMIniT2 digestion test 2
Restriction enzymes:XhoI
Buffer:Fastdigest
Time:10min
7:3009-4pminiT2 (sequenced)
Result:some of them have bands, Lane 4 has band too.
3009pMiniT2 digestion Test 3
1 3009-4 BsrGI +KpnI
2 3009#5 BsrGI +KpnI
3 3009#5 BsrGI +NdeI
4 3009#5 NdeI +NheI
Buffer:NEB 2
Time:30min
Redo 3165 PCR 1st
F1:3028 as template
Primer:3165F1+3165R1
length440bp
extenstion time:1:30
F2:3129 as template
Primer:3165F2+3165R2
Buffer:Platinum
length:165bp
Extension time:15bp
Redo3166 PCR
Primer:3166F+3166R
Template:3017pCEL0
buffer:Platinum
length:60bp
extension time:15s
Date:3/16/2018
[edit]Miniprep of 945_3167
945:1-4 Xba+NcoI Cutsmart 1326
3167:5-8 BsrGI+NheI NEB 2 397
Time:50min
Result:
