456 pLex-CMV-CuO-Dest
Cloning of pLex-CMV-CuO-Dest (456)
Purpose: to clone pLex-CMV-CuO-Dest by PCR
Date: 2011-11-3 to 2011-11-
Notebook: Xiaojing Wang
Page: 004113---004114
Clone ID: 456
Experimenters: Xiaojing Wang
Results
The bands show PCR product of 456,
Primer:
GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG
GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG
Template is 392 pLVB-V5-Dest
Digest result:
digest PCR product and vector with Spe1 + Mlu1,run gel
gel purify, ligate, transform ccdb survival
Colony Screening
Select 5 clones to mini-prep, digestd by Spl I and Mlu I. 456-1,-3.-4 may be correct.
pDonr221_EGFP(393) + pLex-CMV-CuO-Dest(456-1/456-3/456-4) make gateway by LR Clonase II.
Transform 393+456-1/456-3/456-4 by STBL3, Amp.
Select 4 clones each plate to mini-prep. The total number of plasmids is 12 (No.1-12).
No.1-12 plasmids transfect 293T cell line.
48 hours after transfection, take pictures.
No.1-12 plasmids are digested by BamHI. Bands 13 and 14 are control.
Band 13 is 456-3 digested by BamHI, and band 14 is 456-3.
No.1-5,7-9,11,12 may be correct.
Date: 2011-11-7 to 2011-11-10
Notebook: Yingying Lu
Page:
Clone ID: 456
Experimenters: Yingying Lu
Results
GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG
GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG
Template is 392 pLVB-V5-Dest
digest PCR product and vector448pLex-CMV-CuO with Spe1 + Mlu1,run gel
band 1 show insert ,band 2 show vector
5 clonies digest detect:Spe1 + Mlu1, 456-3,456-5 may be right
393EGFP to 456-5 5 clonies transfect into cells
393-pDonr221_EGFP to 456-5 pLex-CMV-CuO-Dest
then digest detect with BamH1
Band 1---5 show 5 clones, band 6 show 456-5 pLex-CMV-CuO-Dest