Yijia LI 3 19 2018
Date:3/19/2018
[edit]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
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
Miniprep of 3166pMiniT2
Restriction enzyme:SalI+BsrGI
Buffer:NEB 3
Time:30min
3129 digestion as vector
Miniprep of 3161
Restriction enzymes:NdeI+XhoI
Buffer:Fastdigest
TIme:30min
Result:combine the rest of them except#2 to do sequence
Redo Subclone
3131:BssHII+XhoI
Buffer:Cutsmart
Results:cut out the middle band of insert, and cut out the vector.
Date:3/20/2018
[edit]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
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)
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
Date:3/21/2018
[edit]Miniprep of 3166(from PCR)
Restriction enzymes:BsrGI+NdeI
Buffer:Fastdigest
Time:30min
Result:#1,#3,#5 have bands
Screening of 3166 miniprep
1,3,5: BsrGI+SalI
1,3,5:BsrGI
Time:15min
Buffer:Fastdigest
Result:good
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
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
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.
