1380 pCMV5-Fc-(3c)-GNE-WPRE-oriP: Difference between revisions
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Latest revision as of 10:58, 25 September 2012
1380 Cloning of pCMV5-Fc-(3c)-GNE-WPRE-oriP
[edit]Purpose: subcloning of gene GNE from 1269 pCMV5-Fc(3c)-GNE into 1369 pCMV5-Fc-(3c)-TagRFP-WPRE-oriP with NheI and NotI
Date: 2012-09-20
Notebook:
Clone ID: 1380
Experimenters: Xiuli
Results
[edit]Cloning
<br>
09-20-2012
A.Restriction enzyme digestion: plasmid 1284,1276 1264, 1269, 1263 and 1396 cut with NheI and NotI.<br>
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<br>
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
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.