3129 pCMV5-HA-VH-mIgG2b
in pMiniT2 vector digestion with Xho1
date:1/31/2018 by Yijia
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
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
| 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 |
.JPG)
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
Crm (talk) 16:55, 3 March 2018 (EST)
Date: 3/3/2018
by Paul
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
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 Iinsert length:527
3129:Kpn I+Not Iinset length:1034
Result:seems good, chose 3129-1 to do sequencing