Paul.xu at 18:02, 20 May 2013

2013-05-20T18:02:53Z

New page

== '''Cloning of 1144 pT7A3-MBP-(3C)-Bst-Exo-''' ==

'''Purpose:''' Cloning of1144 pT7A3-MBP-(3C)-Bst-Exo-

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

'''Notebook:'''

'''Page:'''

'''Clone ID:1144'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== cloning ===

Date:2013-3- 

Gene synthesis 3 PCR fragments

[[Image:1144first round pcr.JPG]]  then use Fragment1,2,3 as template

Bst-Exo-1-1+Bst-Exo-3-18 as primers

[[Image:11442ndround pcr.JPG]] 

digest :

[[Image:Digest for 1144.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
| 
| band 2,3 
| band 4 
|-
| 
|   PCR product 
| 1514 
|-
| enzymes 
| colspan="2" |        Nde1+ Xho1 
|}

 

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

=== Colony Screening ===

Date: 2013

[[Image:1048 1144 1592digest.JPG]]

SEND 1144-3 TO SEQUENCE, MANY GAPS

 

== '''Cloning of 1144 pEC-Bst-Exo-''' ==

'''Purpose:''' Cloning of1144 pEC-Bst-Exo-

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

'''Notebook:'''

'''Page:'''

'''Clone ID:1144'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== cloning ===

Date:2013-3- 

Gene synthesis 3 PCR fragments

[[Image:1144-1 -2 -3PCR.JPG]]

  then use Fragment1,2,3 as template

Bst-Exo-1-1+Bst-Exo-3-18 as primers

[[Image:1144PCR.JPG]] 

digest :

[[Image:Digest for 1144pec.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
| 
| band 1 
| band 3 
|-
| 
|   PCR product 
| 1454 
|-
| enzymes 
| colspan="2" |        Nde1+ Xho1 
|}

 

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

=== Colony Screening ===

Date: 2013

DIGEST WITH NdeI , XhoI and BamHI (should get there bands=>350bp, 1500bp,2500bp)

[[Image:1144pEC-Bst-ExoDIGEST.JPG]] 

SEND 1144-1 TO SEQUENCE, missing 3nt

send 1144-2 to sequence, missing 1G(SEQUENCE FROM T7 TERM AND M13R) 

 

=== cloning ===

Date:2013-3- 

Fragment 1, use Bst-Exo-1-1 and Bst-Exo-3-12

Fragment 2,use Bst-Exo-3-13 and Bst-Exo-3-18 

[[Image:1144fix 1705pcr 2013 04 10.JPG]]  then use Fragment 1and2 as template

Bst-Exo-1-1+Bst-Exo-3-18 as primers

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

digest :

[[Image:Digest for 1144 04 11 2013.JPG]] 

 

=== Colony Screening ===

Date: 2013

DIGEST WITH NdeI , XhoI and BamHI (should get there bands=>350bp, 1500bp,2500bp)

[[Image:1313 1314 1144FIX DIGEST.JPG]][[Image:1144FIX DIGEST.JPG]] 

SEND 1144FIX-5 TO SEQUENCE, MISS ONE T and ONE A,NEED TO FIX THEM 

=== cloning ===

Date:2013-3- 

Fragment 1, use Bst-Exo-1-1 and Bst-Exo-2-8

Fragment 2,use Bst-Exo-2-9 and Bst-Exo-3-18 

[[Image:1144fixthe missing AT using1144fix-5as template.JPG]]  cut out the right bands, then use Fragment 1and2 as template

Bst-Exo-1-1+Bst-Exo-3-18 as primers

[[Image:1144fixthe missing AT.JPG]] 

digest :

[[Image:Digest for1144fixthe missing AT.JPG]] 

 

=== Colony Screening ===

Date: 2013

DIGEST WITH NdeI , XhoI and BamHI (should get there bands=>350bp, 1500bp,2500bp)

[[Image:1144fixthe missing ATDIGEST.JPG]] 

[[Image:562fix_622_1144fix3_1740digest.JPG]]

 

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

* 

== '''Conclusion''' ==

 

 

[[Category:Cloning]]

1144 pT7A3-MBP-(3C)-Bst-Exo- - Revision history

Paul.xu at 18:02, 20 May 2013