Paul.xu at 19:33, 25 May 2012

2012-05-25T19:33:59Z

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Cloning of PfuMBP by Gene synthesis

5/1/2012

PCR 2 fragments, 25 cycles using Phusion or Kapa HiFi, cut out fragments and purify by gel extraction kit.

Join two fragments by Gibson

[[Image:PfuMBP gene synthesis 5 1 2012.jpg]]

mix two PCR fragments, and join them using Gibson Mix

[[Image:1129 gibson reaction2 5 1 2012.JPG]][[Image:1129 gibson reaction 5 1 2012.JPG]]

purify, jointed fragment with Nde1 + Kpn1, cut the top band,  digest pDB.His.MBP with Nde1 + Kpn1, ligate and transform DH5lapha.

5/3/2012: 

 

[[Image:1128 Nde1 Kpn1 digestion 5 3 2012.jpg]]

5/5/2012, make more miniprep digest with Nde1 + Kpn1

digest miniprep with Kpn1+ Nde1. DNA marker is Fermentas 1kb plus (20000, 10000, 7000, '''5000''', 4000, 3000, 2000, '''1500''', 1000, 700, '''500''', 400, 300, 200, 75). size is too big (should be 1180bp) so the top band is not the right product, should use the bottom band.

Select 1128-2 to sequence (result is not good, poor quality) 

====== = ======

'''REPEAT'''

[[Image:1128 digestion 5 4 2012.jpg]]

5/5/2012, repeat gene synthesis and cloning, using Kapa HiFi. 

gene synthesis: 

fragment 1, 10 cycles with 0.25 ul Kapa HiFi without outer primers 

Fragment 2, 10 cycles with 0.25 ul Kapa HiFi without outer primers 

Mix reaction 1 + 2, 20 cycles 

digest gel purified fragment with Nde1 + Kpn1, digest vector 360 with Nde1 + Kpn1 

[[Image:1128 gene synthesis 5 6 2012.jpg]] 

5/6/2012 

[[Image:1128 Nde1 Kpn1 5 6 2012.jpg]] 

ligate 4 hours and transform DH5alpha

1128 PCR Screening 5/7/2012

[[Image:1128 PCR Screening 5.7.2012.JPG]] 

Select 4 clones to mini-prep 

1128 plasmids are digested by Nde1+Kpn1 

[[Image:1128 digested 5.8.2012.JPG]]

sequencing: 2 and 4 have errors

 

== '''Cloning of 1128 pDB.His.PfuMBP-TEV''' ==

'''Purpose:''' Cloning of 1128 pDB.His.PfuMBP-TEV

'''Date: '''2012-5-16---2012-5-22

'''Notebook:0'''

'''Page:'''

'''Clone ID:1128'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1128 PCR 5-17-2012.JPG]] 

1. PCR 5 cycles 0.5 ul of Pfu-MBP1~Pfu-MBP1-16 mix, 5 cycles 0.5 ul Pfu-MBP2-1 ~Pfu-MBP2-16 mix, in 25 ul final volume. 2. Mix reaction 1 + 2, add 1 ul of 10 uM Pfu-MBP1-1 & 1 ul of 10 uM Pfu-MBP2-16, PCR 25 cycles. 

[[Image:1128+1129+1073 clone 5-17-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| 360 pDB.His.MBP 
|-
| enzymes 
| colspan="2" |             Kpn1 + Nde1 
|}

 

2012-5-18

[[Image:1128+1129 clone 5-18-2012.JPG]]

{| cellspacing="1" cellpadding="1" border="1" style="width: 320px; height: 96px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| 360 pDB.His.MBP 
|-
| enzymes 
| colspan="2" |             Kpn1 + Nde1 
|}

 

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

=== Colony Screening ===

Date: 2012-5-21

Make miniprep and digest with Kpn1 + Nde1

[[Image:1128+1129 digest detect 5-21-2012.JPG]] 

clone 2 need to sequence 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

{| cellspacing="1"
|-
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== '''Cloning of 1128 pDB.His.PfuMBP-TEV''' ==

'''Purpose:''' Cloning of1128 pDB.His.PfuMBP-TEV

'''Date: '''2012-5-22---2012-5-25

'''Notebook:0'''

'''Page:'''

'''Clone ID:1128'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1128 PCR 5-23-2012.JPG]] 

Template: 1129 pDB.His.PfuMBP 

1 ul of 10 uM Pfu-MBP1-1 & 1 ul of 10 uM Pfu-MBP2-16, PCR 25 cycles 

[[Image:1128 clone 5-23-2012.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| 360 pDB.His.MBP 
|-
| enzymes 
| colspan="2" |           Kpn1 + Nde1 
|}

 

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

=== Colony Screening ===

Date: 2012-5-25

Make miniprep and digest with Kpn1 + Nde1 

[[Image:1128_digest_detect_5-25-2012.JPG]] 

clone 1,2 may be right

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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

&lt;br&gt;

{| cellspacing="1"
|-
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|}

 

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[[Category:Cloning]]

1128 pDB.His.PfuMBP-TEV - Revision history

Paul.xu at 19:33, 25 May 2012