Editing 3208 pEPD-FW

== '''Cloning of''' ==

'''Purpose:'''&nbsp;&nbsp;

'''Date: '''2018-6-22

'''Notebook:&nbsp;'''

'''Page:'''

'''Clone ID:3208'''

'''Experimenters:'''&nbsp;Yijia, Paul&nbsp;

== '''Results''' ==

=== '''PCR''' ===

Date:&nbsp;Date: 6/20/2018

By: Yijia

template:3152 pCEH0-Flag-VH-mIgG2a &nbsp;;3010 pCMV5-Flag-kappa as template

primers:

Fragment 1: PCR from 3152 pCEH0-Flag-VH-mIgG2a as template

3208-F1 ACTCGCGGCCCAGCCGGCCATGgctgtacactccGAGGTTCAGCTGCAGCAGTC

3208-R1 aGATCCGCCACCGCCGGATCCACCTCCGCCTGAACCGCCTCCACCGCTAGCAGAGGACACGGTCAGTGTG (CACACTGACCGTGTCCTCTGCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCt)

Fragment 2: PCR from 3010 pCMV5-Flag-kappa as template

3208-F2 GATCCGGCGGTGGCGGATCt aga tct GCGCGC TGTGACGTGCTGATGACACAGGCTC

3208-R2 atggtgttggcctcccgggccTAGGACCtcgAGcttGgtAcctccgccaaaggTG



Gel:

'''1st PCR of 3208'''

[[File:IMG 0915.JPG|RTENOTITLE]]

'''2nd PCR of 3208'''

[[File:IMG 0922.JPG|RTENOTITLE]]



'''Cloning'''

By: Paul

Vector: 3200 pEPD digested with SfiI

Ligation: 30 min with T4 DNA ligase

Competent cells: NEBTurbo



'''Colony PCR Screening&nbsp;'''



pADL-F and 3208-R2

'''[[File:3208 colony screening.JPG|RTENOTITLE]]'''



'''Restriction Digestion'''

Date: 6/21/2018

By: Paul

Nco1 + Xho1

[[File:3208 digestion1.JPG|RTENOTITLE]]

[[File:3208 3231 3230 digestion.JPG|RTENOTITLE]]

'''Sequencing'''

6/22/18, clone #1, no good, missing 1C

pick more clones, make moiniprep.

[[File:3208 digestion2.JPG|RTENOTITLE]]

send #8 to sequencing

sequencing: clone #8 is good

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

== '''Conclusion''' ==
[[Category:Cloning]]