1377 pCMV5-Fc-(3c)-NPR1-Cyto-WPRE-oriP: Difference between revisions
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Latest revision as of 11:07, 25 September 2012
Cloning of pCMV5-Fc-(3c)-NPR1-Cyto-WPRE-oriP
[edit]Purpose: subcloning of gene NPR1-cyto from 1284 pCMV5-Fc(3c)-NPR1-Cyto into 1369 pCMV5-Fc-(3c)-TagRFP-WPRE-oriP with NheI and NotI
Date: 2012-09-20
Notebook: 
Page:
Clone ID: 1377
Experimenters: Xiuli
insert size: ~ 1647bp
Results
[edit]
Cloning
09-19-2012
A.Restriction enzyme digestion: plasmid 1284,1276 1264, 1269, 1263 and 1396 cut with NheI and NotI.
B. Gel purification and elute with 15 ul H2O
c Ligation: 2xLigation buffer (12ul) +12 ul elution from above +0,5 ul T7 ligase. Incubate at RT for 1 hour
D. Transformation.
9/22/2012
make miniprep, digest with Nhe1 + Not1
9/24/2012
Select 1377-2, 1378-1, 1379-2, 1380-2, 1381-1 to make maxiprep using Qiagen maxiprep kit. 1381, 1392 using invitrogen maxiprep kit
1377-1378-1379-1380-1380-1381 Plasmid Maxiprep
Clones for maxiprep
| Clone ID |
Parent |
Clone Name |
Expected Size (bp) |
Actual Size on Gel (Kb) |
Clone Identity |
DNA conc |
OD260/280 |
RNA |
| 1377-1 |
1284 |
1647 |
2.5 |
|||||
| 1378-2 |
1276 |
1322 |
1.6 |
|||||
| 1379-2 |
1264 |
3205 |
4 |
|||||
| 1380-2 |
1269 |
2169 |
3 |
|||||
| 1381-1 |
1263 |
1586 |
2.5 |
1381 and 1392 using Invitrogen maxi prep kit have too much RNA. After treating sample 1381 and 1392 with 0.5 ul RNAse (Qiagen) at 37oC for 2-3 hours, precipitate DNA with isoprapanol.