Paul.xu at 13:14, 8 May 2012

2012-05-08T13:14:10Z

New page

== '''Cloning of 1090-pCMVK-RFP-Fc''' ==

'''Purpose:''' Cloning of 1090-pCMVK-RFP-Fc

'''Date: '''2012-4-20----2010-4-23

'''Notebook:0'''

'''Page:'''

'''Clone ID:1090'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

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. 

[[Image:1090 clone 4-20-2012-2.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 459px; height: 82px;"
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| band 2 
| band 1 
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| Gibson mixture 
| 380 pCMVK-IL2-intFc-IRES-TagGFP. 
|-
| enzymes 
| colspan="2" |                       BglII + Sal1 
|}

 

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=== Colony Screening ===

Date: 2012-4-23

Make miniprep and digest with BglII + Sal1 

[[Image:1090 digest detect 4-23-2012.JPG]] 

not the right band ,only the origin of vector

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

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== '''Cloning of 1090-pCMVK-RFP-Fc''' ==

'''Purpose:''' Cloning of 1090-pCMVK-RFP-Fc

'''Date: '''2012-4-25----2010-4-30

'''Notebook:0'''

'''Page:'''

'''Clone ID:1090'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

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

[[Image:1090 Gibson mix 4-25-2012.JPG]] 4. Digest with BglII + Sal1. 5. Ligate to BglII + Sal1 digested pCMVK-IL2-intFc-IRES-TagGFP. 

[[Image:1068+1070+1090+1092 clone 4-26-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 459px; height: 82px;"
|-
| 
| band 5 
| band 6 
|-
| 
| Gibson mixture 
| 380 pCMVK-IL2-intFc-IRES-TagGFP. 
|-
| enzymes 
| colspan="2" |                       BglII + Sal1 
|}

 

{| cellspacing="1" cellpadding="1" border="1"
|-
| 
|}

=== Colony Screening ===

Date: 2012-4-30

Make miniprep and digest with BglII + Sal1 

[[Image:1070+1090 digest detect 4-30-2012.JPG]]

need to transfection and sequence

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

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== '''Cloning of 1090-pCMVK-RFP-Fc''' ==

'''Purpose:''' Cloning of 1090-pCMVK-RFP-Fc

'''Date: '''2012-5-8----20

'''Notebook:0'''

'''Page:'''

'''Clone ID:1090'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

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

[[Image:1090_P1+P2_4-25-2012.JPG]][[Image:1090_PCR_5-8-2012.JPG]]

 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 459px; height: 82px;"
|-
| 
| band 2 
| band 1 
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| 
| 
| 3 
|-
| enzymes 
| colspan="2" |                 
|}

 

{| cellspacing="1" cellpadding="1" border="1"
|-
| 
|}

=== Colony Screening ===

Date: 2012-

Make miniprep and digest with 

not the right band ,only the origin of vector

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

 

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[[Category:Cloning]]

1090-pCMVK-RFP-Fc - Revision history

Paul.xu at 13:14, 8 May 2012