947 pT7A30-6His-pMBP
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
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.
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
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.
Date:3/15/2018
[edit]Subclone of 947 Vector digstion Time:30min
Buffer:cutsmart
Vecotor:1727
XbaI+NcoI
Result:good, cut out and purify
Date:3/19/2018
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
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
Date:2/26/2018
Test of 947_945_3166_3165
| Name | Enzymes | Size (bp) | Lane |
| 947 | BglII | 646 | 1,2,3 |
| BssHII | 1856 | 4,5,6 | |
| 945 | BglII | 628 | 11,12 |
| BssHII | 1838 | 13 | |
| 3165 | SalI+BsrGI | 693 | 8 |
| HindIII+BsrGI | 441 | 10 | |
| 3166 | SalI+BsrGI | 1234 | 9 |
| XbaI | 2049 | 7 | |
| BglII | 3259 | to do |
Date:4/2/2018
[edit]redo Subclone of 947
Vector:1727
Insert:947pMiniT
Size:1344 bp
Date:4/4/2018
[edit]miniprep digestion of 984_947_3152
- Name Enzymes Size:
1-4:984 XbaI+NheI 957bp
5 :982(973-1pMiniT) XbaI+NheI
6-9:947 XbaI+NcoI-HF 1344bp
10:947pMiniT XbaI+NcoI-HF
11:947(from PCR cloning) XbaI+NcoI-HF
12-15:3152 BsrGI+NheI 371bp
16: 3162(Vector) BsrGI+NheI
