382 pCMVK-IRES-TagGFP
Cloning of pCMVK-IRES-TagGFP
Purpose: to clone pCMVK-IRES-TagGFP by PCR
Date: 2011-9-14---9-17
Notebook:Lu yingying
Page:003896---003903
Clone ID:382
Experimenters: lu yingying
Results
[edit]
All bands show PCR product of IRES-TagGFP,
template:TagGFP-F3R3-PET28b(2)
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
Digest result:Xho1+BglII
band1,2,3 show PCMVK vector, band 4,5,6 show IRES-TagGFP
Gene Synthesis
Colony Screening
Result: no GFP fluorescence when transfected into cells.
Restart:
Date: 2011-9-23---
Notebook:Lu yingying
Page:003911---
Clone ID:382
Experimenters: lu yingying
Results
bands show 1st round PCR product
template with gene synthesis method:TagGFP oligo mixture of TagGFP1 to TagGFP 20
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
25 cycles
bands show 2nd round PCR product
1st round PCR product as template,
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
20 cycles
Gene Synthesis
Colony Screening
Result: no GFP fluorescence when transfected into cells.
Restart:
Date: 2011-9-26---
Notebook:Lu yingying
Page:00---
Clone ID:382
Experimenters: lu yingying
Result:
band1,2,3
template:TagGFP-F3R3-PET28b(2)
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
25 cycles
band 4,5,6
template with gene synthesis method:TagGFP oligo mixture of TagGFP1 to TagGFP 20
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
25 cycles
digest :Xho1+BglII
digest :Xho1+BglII
restart
Date: 2011-9-27---
Notebook:Lu yingying
Page:00---
Clone ID:382
Experimenters: lu yingying
Result
band1,2,3,4
template:TagGFP-F3R3-PET28b(4)
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
25 cycles
band 6,7 show TagGFP digest result
band 8 shows PCMVK digest result
Xho1+BglII
Result: only two clones in plate
restart
Date: 2011-9-28---
Notebook:Lu yingying
Page:00---
Clone ID:382
Experimenters: lu yingying
Result
band1,2,3,4
template:TagGFP-F3R3-PET28b(4)
Primer-F 8685-TagGFP-F Xho1 HindIII 22nt IRES Kozak TagGFP atcg CTCGAG AAGCTT TTTATAATTTCTTCTTCCAGAA GCCACC ATG AGC GGG GGC GAG
Primer-R 8685-TagGFP-R BglII (CATGGACGAGCTGTACAGG taa AGATCT atgct) agcatAGATCTttaCCTGTACAGCTCGTCCATG
25 cycles
bands show digest result,digest :Xho1+BglII
TagGFP-PCMVK digest result:Xho1+BglII
upper bands show PCMVK vector
lower bands show TagGFP
Transfection Result:
[edit]clone 2 ,3 , 4 have green fluorescence
--Luyingying 14:31, 1 October 2011 (EST)