1090-pCMVK-RFP-Fc
Cloning of 1090-pCMVK-RFP-Fc
[edit]Purpose: Cloning of 1090-pCMVK-RFP-Fc
Date: 2012-4-20----2010-4-23
Notebook:0
Page:
Clone ID:1090
Experimenters: yingying
Results
[edit]cloning
[edit]Date:2012-4-20
RFP-Fc-Sal1-F1
accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATC
RFP-Fc-Sal1-F2
CTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG
RFP-Fc-R1
(CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC)
GTGGGCATGTGTGAGTTTTGTCGCCGCTGCCAGTTTGTGCCCCAGTTTGCTAG
RFP-Fc-F3
CTAGCAAACTGGGGCACAAACTGGCAGCGGCGACAAAACTCACACATGCCCAC
RFP-Fc-R2
(CTCTCCCTGTCTCCGGGTAAAGAGAACCTGTACTTCCAGGGCgattacaaggatgacgatg)
catcgtcatccttgtaatcGCCCTGGAAGTACAGGTTCTCTTTACCCGGAGACAGGGAGAG
RFP-Fc-F4
CAGGGCgattacaaggatgacgatg GAa ttC ACT AGT CTG CAG AAG CTT ctc gag CAT CAC
RFP-Fc-BglII-R3
(CAG AAG CTT ctc gag CAT CAC CAT CAC CAT CAC TGA ACGCGT TTAATTAA agatct aagctt)
aagcttagatctTTAATTAAACGCGTTCAGTGATGGTGATGGTGATGctcgagAAGCTTCTG
How to make
1. PCR using RFP-Fc-Sal1-F1, 10% RFP-Fc-Sal1-F2 + RFP-Fc-R1 using 114 pET28b-TagRFP as template --> to amplify RFP
2. PCR using RFP-Fc-F3, + 10% RFP-Fc-R2, 10% RFP-Fc-F4 + RFP-Fc-BglII-R3 and 380 pCMVK-IL2-intFc-IRES-TagGFP as template,
to amplify Fc domain.
3. Join two fragment using Gibson mix, gel purify
4. Digest with BglII + Sal1.
5. Ligate to BglII + Sal1 digested pCMVK-IL2-intFc-IRES-TagGFP.
| band 2 |
band 1 | |
| Gibson mixture |
380 pCMVK-IL2-intFc-IRES-TagGFP. | |
| enzymes |
BglII + Sal1 | |
Colony Screening
[edit]Date: 2012-4-23
Make miniprep and digest with BglII + Sal1
not the right band ,only the origin of vector
Result intepretation/Summary
[edit]Conclusion
[edit]
<br>
Cloning of 1090-pCMVK-RFP-Fc
[edit]Purpose: Cloning of 1090-pCMVK-RFP-Fc
Date: 2012-4-25----2010-4-30
Notebook:0
Page:
Clone ID:1090
Experimenters: yingying
Results
[edit]cloning
[edit]Date:2012-4-25
RFP-Fc-Sal1-F1
accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATC
RFP-Fc-Sal1-F2
CTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG
RFP-Fc-R1
(CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC)
GTGGGCATGTGTGAGTTTTGTCGCCGCTGCCAGTTTGTGCCCCAGTTTGCTAG
RFP-Fc-F3
CTAGCAAACTGGGGCACAAACTGGCAGCGGCGACAAAACTCACACATGCCCAC
RFP-Fc-R2
(CTCTCCCTGTCTCCGGGTAAAGAGAACCTGTACTTCCAGGGCgattacaaggatgacgatg)
catcgtcatccttgtaatcGCCCTGGAAGTACAGGTTCTCTTTACCCGGAGACAGGGAGAG
RFP-Fc-F4
CAGGGCgattacaaggatgacgatg GAa ttC ACT AGT CTG CAG AAG CTT ctc gag CAT CAC
RFP-Fc-BglII-R3
(CAG AAG CTT ctc gag CAT CAC CAT CAC CAT CAC TGA ACGCGT TTAATTAA agatct aagctt)
aagcttagatctTTAATTAAACGCGTTCAGTGATGGTGATGGTGATGctcgagAAGCTTCTG
How to make
1. PCR using RFP-Fc-Sal1-F1, 10% RFP-Fc-Sal1-F2 + RFP-Fc-R1 using 114 pET28b-TagRFP as template --> to amplify RFP
2. PCR using RFP-Fc-F3, + 10% RFP-Fc-R2, 10% RFP-Fc-F4 + RFP-Fc-BglII-R3 and 380 pCMVK-IL2-intFc-IRES-TagGFP as template,
to amplify Fc domain.
3. Join two fragment using Gibson mix, gel purify
4. Digest with BglII + Sal1.
5. Ligate to BglII + Sal1 digested pCMVK-IL2-intFc-IRES-TagGFP.
| band 5 |
band 6 | |
| Gibson mixture |
380 pCMVK-IL2-intFc-IRES-TagGFP. | |
| enzymes |
BglII + Sal1 | |
Colony Screening
[edit]Date: 2012-4-30
Make miniprep and digest with BglII + Sal1
need to transfection and sequence
Result intepretation/Summary
[edit]Conclusion
[edit]
<br>
Cloning of 1090-pCMVK-RFP-Fc
[edit]Purpose: Cloning of 1090-pCMVK-RFP-Fc
Date: 2012-5-8----20
Notebook:0
Page:
Clone ID:1090
Experimenters: yingying
Results
[edit]cloning
[edit]Date:2012-5-8
RFP-Fc-Sal1-F1
accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATC
RFP-Fc-Sal1-F2
CTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG
RFP-Fc-R1
(CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC)
GTGGGCATGTGTGAGTTTTGTCGCCGCTGCCAGTTTGTGCCCCAGTTTGCTAG
RFP-Fc-F3
CTAGCAAACTGGGGCACAAACTGGCAGCGGCGACAAAACTCACACATGCCCAC
RFP-Fc-R2
(CTCTCCCTGTCTCCGGGTAAAGAGAACCTGTACTTCCAGGGCgattacaaggatgacgatg)
catcgtcatccttgtaatcGCCCTGGAAGTACAGGTTCTCTTTACCCGGAGACAGGGAGAG
RFP-Fc-F4
CAGGGCgattacaaggatgacgatg GAa ttC ACT AGT CTG CAG AAG CTT ctc gag CAT CAC
RFP-Fc-BglII-R3
(CAG AAG CTT ctc gag CAT CAC CAT CAC CAT CAC TGA ACGCGT TTAATTAA agatct aagctt)
aagcttagatctTTAATTAAACGCGTTCAGTGATGGTGATGGTGATGctcgagAAGCTTCTG
How to make
1. PCR1 using RFP-Fc-Sal1-F1, 10% RFP-Fc-Sal1-F2 + RFP-Fc-R1 using 114 pET28b-TagRFP as template --> to amplify RFP
2. PCR2 using RFP-Fc-F3, + 10% RFP-Fc-R2, 10% RFP-Fc-F4 + RFP-Fc-BglII-R3 and 380 pCMVK-IL2-intFc-IRES-TagGFP as template,---> to amplify Fc domain.
3. PCR3 using PCR1+PCR2 as template,RFP-Fc-Sal1-F1+ RFP-Fc-BglII-R3 as primers
| band 2 |
band 1 | |
| 3 | ||
| enzymes |
| |
Colony Screening
[edit]Date: 2012-
Make miniprep and digest with
not the right band ,only the origin of vector
Result intepretation/Summary
[edit]Conclusion
[edit]
<br>