Editing 3122 pET30b-CRM197

in pJet1.2

[[File:938 939 942 3122 Nde1 Nco1 Xho1.JPG|RTENOTITLE]]

[[File:938 939 942 3122 Xho1 HindIII.JPG|RTENOTITLE]]--[[User:Paul.xu|Paul.xu]] ([[User_talk:Paul.xu|talk]]) 09:04, 21 December 2017 (EST)

12/27/2017

938 digestion with Nde1-Xho1, Fastdigest Buffer

[[File:938 939 942 3122 931 1855 digestion.JPG|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width: 681px;"
|-
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| style="width: 54px;" | 8,9
| style="width: 55px;" | 10,11
| style="width: 71px;" | 13
|-
| 938&nbsp;PNGase_F
| <span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">939 Tte-ExoIII</span><br/>
| 942 VVN<br/>
| 3122 CRM197<br/>
| 931, 1, PAK16D ligase&nbsp;<br/>
| 931, 2, PAK16D ligase<br/>
| 1855&nbsp; yPolyA-Pol<br/>
| style="width: 54px;" | 1540
| style="width: 55px;" | 2046
| style="width: 71px;" | 2046<br/>
|-
| Nde1-Xho1
| Nde1-Xho1<br/>
| <span style="color:#FF0000;">Nco1-</span>Xho1<br/>
| Nde1-Xho1<br/>
| Nde1-<span style="color:#FF0000;">HindIII</span><br/>
| <span style="color:#FF0000;">HindIII</span>-Xho1
| Nde1-Xho1
| style="width: 54px;" | Nde1-Xho1
| style="width: 55px;" | Nde1-Xho1
| style="width: 71px;" | <span style="color:#FF0000;">Nco1</span>-Xho1
|-
| pJet1.2
| pJet1.2<br/>
| pJet1.2<br/>
| pJet1.2<br/>
| pJet1.2<br/>
| pJet1.2<br/>
| pEC2-<br/>
| style="width: 54px;" | pET30b
| style="width: 55px;" | pET28b
| style="width: 71px;" | pET28b<br/>
|-
| 1145<br/>
| 780<br/>
| 716<br/>
| 1798<br/>
| 1174<br/>
| 986<br/>
| 1713
| style="width: 54px;" | there is no Xho1 site
| style="width: 55px;" | 1377
| style="width: 71px;" | ~1377
|-
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
| style="width: 54px;" | <br/>
| style="width: 55px;" | <br/>
| style="width: 71px;" | <br/>
|}

[[File:938 939 942 3122 931 1855 digestion Cutsmart.JPG|RTENOTITLE]]

== Date:3/30/2018 ==

'''Miniprep digestion of 3122_938_939'''

3122:lane 1-4 lane 5&nbsp;:+ size:1743 bp

938: lane 6-9 lane 10:+ size:1145 bp

939: lane 11-14 lane 15:+ size:780 bp

Positive control are insert-pjet1.2

The small band size of vector 1727:193 bp

[[File:IMG 0535.JPG|RTENOTITLE]]

'''redo Miniprep digestion of 3122_938_939'''

1:3122&nbsp; size:1743bp

2:3122pjet1.2 sequenced +

3:938&nbsp; size:1145 bp

4:938pjet1.2 sequenced +

5,6,7:939&nbsp; size:780 bp

8:939pjet1.2 sequenced +

Enzymes:NdeI+XhoI

[[File:IMG 0536.JPG|RTENOTITLE]]

== Date:4/5/2018 ==

'''824subclone'''

Insert size:1743bp

Vector:1987pT7A3

[[File:Subclone3122.JPG|RTENOTITLE]]

=== Date:5/29/2018 ===

'''Subclone and Vector digestion'''

[[File:IMG 0860.JPG|RTENOTITLE]]

3122: what is the vector name? clone #500 , insert size should be 2055 (Nde1 + Xho1)

'''Miniprep digestion of 2503_3122_3197_3191'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| lane
| Enzymes
| Size
| Note
|-
| 2503
| 1-4
| Nde1+Xho1
| 951bp
| #1,2 seems good
|-
| 3122
| 5-8
| Nde1+Xho1
| 1743bp
| #5,6,8 seems good
|-
| 3197
| 9-12
| BamH1+Nco1
| 1 band
| may not good
|-
| 3191
| 13-16
| BamH1+Nco1
| 722bp
| #14,15,16 seems good
|}

[[File:IMG 0872.JPG|RTENOTITLE]]

3122 could be parental vector

'''Miniprep of 3122_3210'''

[[File:IMG 0902.JPG|RTENOTITLE]]

'''Miniprep of 3122'''

[[File:IMG 0926.JPG|RTENOTITLE]]&nbsp;
[[Category:Cloning]]