Paul.xu at 19:42, 25 April 2013

2013-04-25T19:42:29Z

New page

== '''Cloning of 1048-pEC-RNaseI''' ==

'''Purpose:''' Cloning of1048-pEC-RNaseI

'''Date: '''2013-3-

'''Notebook:'''

'''Page:'''

'''Clone ID:1048'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== cloning ===

Date:2013-3- 

 gene synthesis 

Fragment1: RnaseI-1 ~RnaseI-20, ~650 bp, Fragment 2: RnaseI-21~40, ~740 bp, 

[[Image:1048-1 1048-2 1535pcr.JPG]]  then use Fragment1+Fragment 2 as template

RnaseI-1+RnaseI-40 as primers

[[Image:1048pcr.JPG]] 

digest :

[[Image:Digest for 1048.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
| 
| band 2,3 
| band 4 
|-
| 
|   PCR product 
| pEC 
|-
| enzymes 
| colspan="2" |        Nco1+ Xho1 
|}

 

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

=== Colony Screening ===

Date: 2013

[[Image:1048digest.JPG]]

send 1048-1 to sequence, has a G to T mutation and frame shift,  need to use use oligo to fix the frame shift and the mutation. 

=== cloning ===

pcr 

Fragment1: use RnaseI-Nco1-F and RnaseI-18 as primer Fragment 2: use RnaseI-19 and RnaseI-40 as primer 

[[Image:1144fix 1048fix 2013 04 11.JPG]] 

then use Fragment1+Fragment 2 as template

RnaseI-1+RnaseI-40 as primers

[[Image:1048fix 2013 04 11.JPG]] 

 

digest :

[[Image:Digest for 1048for fix the frame shift.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
| 
| band 1 
| band3 
|-
| 
| pEC 
| PCR product 
|-
| enzymes 
| colspan="2" |        Nco1+ Xho1 
|}

 

=== Colony Screening ===

Date: 2013

[[Image:1048fixdigest_04_25_2013.JPG‎]]

 

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

* 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1048-pEC-RNaseI - Revision history

Paul.xu at 19:42, 25 April 2013