Crm at 23:20, 13 March 2018

2018-03-13T23:20:17Z

New page

in pMiniT2 vector digestion with Xho1

date:1/31/2018 by Yijia

[[File:3130 3129 3021 3075.JPG|RTENOTITLE]]

2/4/2018

3129 pCMV5-HA-VH-mIgG2b, heavy Vector Name: 2492 pCMV5-mIgG2b-HC

finished: PCR from  3011 pJet1.2-HA Heavy chain

 3129-BamH1-F atcatcggatccAAGATCTAGAGCCGCCACCATG

3129-Kpn1-F atcatcGTACCagacacggtaaccagtgttc (gaacactggttaccgtgtctGGTACgatgat) Clone into pMiniT2

Then subclone into 2492 pCMV5-mIgG2b-HC Restriction Enzymes: BamH1 - Kpn1

[[File:3001 3076 3010 3129 3130.JPG|RTENOTITLE]]

Date:2/6/2018

PCR Cloning of '''3129'''

'''Figure despriction''' Template: 3011 H in pJet1.2, sequenced, annealing: 55 oC 30'', Extension 72 oC 30''

{| border="1" cellspacing="1" cellpadding="1" style="font-size: 14px; color: rgb(37, 37, 37); font-family: sans-serif; background-color: rgb(255, 255, 255); width: 500px;"
|-
| Lane
| Primers
| Enzyme
|-
| Ladder
| 
|
|-
| 1
| 3129-F + 3129-R 
| DreamTaq 
|-
| 2
| pJet1.2 F + R
| DreamTaq
|-
| 3
| 3129-F + 3129-R 
| Plantimum PCR Supermix HF 
|-
| 4
| pJet1.2 F + R 
| Plantimum PCR Supermix HF 
|-
| 5
| 3129-F + 3129-R 
| Q5 DNA polymerase
|-
| 6
| pJet1.2 F + R 
| Q5 DNA polymerase 
|}

[[File:PCR for 3011(Paul).JPG|RTENOTITLE]]

[[File:3129 digestion test.JPG|RTENOTITLE]]

1:Sac I +BsrG I 2:Sall+BsrG I 3:Xho I+ BsrG I 4:BsrG I 5:Kpn I+BsrG I Volume of DNA:1 ul Buffer:Fastdigest  Time:30mins

[[File:Digestion test for 3129.JPG|RTENOTITLE]]

[[User:Crm|Crm]] ([[User_talk:Crm|talk]]) 16:55, 3 March 2018 (EST)

Date: 3/3/2018

by Paul

[[File:3129 BsrG1 Kpn1 Sal1.JPG|RTENOTITLE]]

Sal1---BsrG1---BsrG1--Kpn1 ?

3129 plasmid digested with

1: BsrG1, 

2: BrsG1+ Kpn1 + Sal1

3, Kpn1+ Sal1

3/4/2018, sample #3, cut out both samll and large fragments, purify with spin column, digest with BsrG1

[[File:3129 Kpn1 Sal1 BsrG1.JPG|RTENOTITLE]]

conclusion: it is most likely there are two BsrG1 sites within Sal1-Kpn1 fragment

Miniprep digestion for 3154_3129

Digestion buffer:Fastdigest

Restriction enzymes:

3154:BamH I+ Kpn I'''insert length:527'''

3129:Kpn I+Not I'''inset length:1034'''

[[File:Miniprep digestion for 3154 3129.JPG|RTENOTITLE]]

Result:seems good, chose 3129-1 to do sequencing
[[Category:Pages with broken file links]] [[Category:Cloning]]

3129 pCMV5-HA-VH-mIgG2b - Revision history

Crm at 23:20, 13 March 2018