Paul.xu at 19:41, 2 April 2014

2014-04-02T19:41:48Z

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== '''Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP''' ==

'''Purpose:''' Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP

'''Date: '''2014-2-3---2014-2-6

'''Notebook:0'''

'''Page:'''

'''Clone ID:2014'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:718 2012 2014 2040 2045 2060clone 1-31-2014.JPG]]
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{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 5 
| 6 
|-
| 
| 1955 
| 2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGFP 
|-
| 
| colspan="2" | BamH1 + Xho1 
|}

 

[[Image:2014clone 2-5-2014.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 1 
| 2 
|-
| 
| pcr 
| 2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGF 
|-
| 
| colspan="2" | BamH1 + Xho1 
|}

 

 

=== Colony Screening ===

Date: 2014-2-6

Make miniprep and digest BamH1+xba1 

[[Image:2014digest 2-6-2014.JPG]] 

c2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGF

 

== '''Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP''' ==

'''Purpose:''' Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP

'''Date: '''2014-2-

'''Notebook:0'''

'''Page:'''

'''Clone ID:2014'''

'''Experimenters:''' shanyi

== '''Results''' ==

=== cloning ===

[[Image:Digest for 2014.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 
| 
|-
| 
| 1955 
| 2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGFP 
|-
| 
| colspan="2" | BamH1 + Xho1 
|}

 

=== Colony Screening ===

Date: 2014-2-6

Make miniprep 

[[Image:2014without digestion.JPG]] 

 digest BamH1+Xho1 

[[Image:2014digest with bx.JPG]] 

digest with XbaI 

for 2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGF there are three xbaI at 630bp, 1985bp(cleavage affected by meth), 2518bp, so digesting with XbaI can get two bands 

for 2014 there is only one XhaI at 1988bp(cleavage affected by meth) 

[[Image:2014digest with b.JPG]] 

SENT 2014-1 TO SEQUENCE WITH CMV-SEQ-F

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

== '''Conclusion''' ==

 

== '''Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP''' ==

'''Purpose:''' Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP

'''Date: '''2014-2-24

'''Notebook:0'''

'''Page:'''

'''Clone ID:2014'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:2012 2014 2015 2076 2078PCR 2-24-2014.JPG]] 

PCR using clone1955  as template LoxP-TurboRFP-BamH1-F3 

TurboGFP-HindIII-Sal1-R 

[[Image:2014clone 2-24-2014.JPG]]

{| width="286" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
|-
| 
| pcr 
| 2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGFP 
|-
| 
| colspan="2" | BamH1 + HindIII 
|}

 

=== Colony Screening ===

Date: 201

Make miniprep and digest with

 

 

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

== '''Conclusion''' ==

== '''Cloning of 2029 pEC/PEC2-LoxP-TurboRFP-ODC-2Stops-LoxP''' ==

'''Purpose:''' Cloning of 2029pEC/PEC2-LoxP-TurboRFP-ODC-2Stops-LoxP

'''Date: '''2014-3-7--2014-3-10

'''Notebook:0'''

'''Page:'''

'''Clone ID:2029'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:2014clone 3-7-2014.JPG]] 

{| width="301" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
| 3 
| 4 
|-
| 
| PCR 
| 1454pEC 
| PCR 
| 2015pEC2 
|-
| 
| colspan="4" | BamH1 + Xho1
|}

 

 

=== Colony Screening ===

Date: 2014-3-10

Make miniprep and digest with  BamH1 + Xho1

[[Image:2014digest 3-10-2014.JPG]] 

1-4:pEC vector 

5-8:pEC2 vector

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

== '''Conclusion''' ==

== '''sub Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP''' ==

'''Purpose:''' Cloning of 2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP

'''Date: '''2014-3-11-2014-3-12

'''Notebook:0'''

'''Page:'''

'''Clone ID:2014'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:2014clone 3-11-2014.JPG]] 

{| width="327" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
|-
| 
| 2029pEC-LoxP-TurboRFP-ODC-2Stops-LoxP 
| 2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGF 
|-
| 
| colspan="2" | BamH1 + Xho1 
|}

 

 

 

=== Colony Screening ===

Date: 2014-3-12

Make miniprep and digest with  

[[Image:2014digest 3-12-2014.JPG]] 

[[Image:2014-7digest 3-12-2014.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
| 3 
|-
| 
| 2014-7 
| 2014-7 
| 2013,2013 pCMV-FRT-TurboRFP-ODC-2Stop-FRT-TurboGF 
|-
| 
| BamH1+Xho1 
| BamH1+Xba1 
| BamH1+Xba1 
|}

 

2014-3-24 

BamH1+Xho1 

[[Image:2014digest 3-24-2014.JPG]] 

There is no control for this digestion, you  should digest with BamH1 alone, Xho1 alone and no digestion for at least one clone. Clone 1, and 2, 4 are not right. Clone #3 also looks weird. You MUST run a few controls  if an experiment does not look right in the future. 

 

2014-4-2

2014 transform DH5a and NEBturbo ,miniprep and digest with BamH1+Xho1/BamH1/Xho1 

[[Image:2014digest 4-2-2014.JPG]]

{| width="300" height="66" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7clone1 
| 8clone1 
|-
| 
| colspan="4" | DH5a 
| colspan="2" | NEBturbo 
| colspan="2" | clone1 
|-
| 
| colspan="6" | BamH1+Xho1 
| BamH1 
| Xho1 
|}

 

2014-clone 1 digest with BamH1+EcoR1/EcoR1+Xho1 

[[Image:2014-1digest_4-2-2014.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
|-
| 
| colspan="2" |    2014-clone 1 
|-
| 
| BamH1+EcoR1 
| EcoR1+Xho1 
|}

 

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

== '''Conclusion''' ==

[[Category:Cloning]]

2014 pCMV-LoxP-TurboRFP-ODC-2Stops-LoxP-TurboGFP - Revision history

Paul.xu at 19:41, 2 April 2014