945 pT7A30-pMBP
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.
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.
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
[[File:Subclone of 945.JPG|RTENOTITLE]
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
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: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 |
945 subclone
Size:1326 bp
Enzymes:Xba1+Nco1
Miniprep digestion of 945_986_944_3127
| # | Lane | Size | Enzymes | Note |
| 945 | 1-4 | 1326bp | Xba1-Nco1 |
subclone |
| 986 | 5-8 | 774bp | HindIII+XhoI | ~4kbp |
| 944 | 9-12 | 2334bp | NdeI+XhoI | PCR |
| 3127 | 13-16 | 402bp | Nde1-HindIII |
Gibson Mix |
