2115 pT7A3-iFLP
Cloning of 2115 pT7A3-iFLP
[edit]Purpose: Cloning of 2115 pT7A3-iFLP==
Date: 2014-3----
Notebook:0
Page:
Clone ID:2115
Experimenters: shanyi
Results
[edit]CLONING
[edit]subclone from 2114 pEC-iFLP to 1860 pT7A3-yFLP using HindIII + Xho1
Colony Screening
[edit]Date: 2014
Make miniprep, digest with NdeI + HindIII + Xho1,there is no NdeI site in new FLP
Result intepretation/Summary
[edit]Conclusion
[edit]Protein Expression
[edit]
| Lane | ID | Protein Name | MW, pI | Tag | Host | Condition | Expression |
| 1 | 927 | pET28b-Cid1 |
42 | His | Nico21(DE3), pLysN | 15 ml, RM , 0.3M IPTG induce o/n | Yes |
| 2 | 3097 | pET28b-pQbeta |
no | Nico21(DE3), pLysN |
15 ml, RM , 0.3M IPTG RM induce o/n |
do not know | |
| 3 | 3098 | pET28b-AP205 |
no | Nico21(DE3), pLysN |
15 ml, RM , 0.3M IPTG RM induce o/n |
do not know | |
| 4 | 3074 | pET28b-HA | no | Nico21(DE3), pLysN |
15 ml, RM , 0.3M IPTG RM induce o/n |
do not know | |
| 5 | 3105 | pET28b-AaLS |
22 | His | Nico21(DE3), pLysN |
15 ml, RM , 0.3M IPTG RM induce o/n |
Yes |
| 6 | M | 198, 98(Y), 62, 49, 38, 28, 17(Red), 14, 6, 3 | |||||
| 7 | 2106 | pET28b-iFLP, KJE7 | 9.76 / 52.8 |
N-His | Tuner(DE3) |
15 ml, Rm, 0.3M IPTG RM induce o/n | |
| 8 | 2115 | pT7A3-iFLP, KJE7 |
9.76 / 52.8 |
N-His |
Tuner(DE3) |
15 ml, Rm, 0.3M IPTG RM induce o/n |
|
| 9 | 2106 | pET28b-iFLP, KJE8 |
9.76 / 52.8 |
N-His |
Tuner(DE3) | 15 ml, Rm, 0.3M IPTG RM induce o/n |
|
| 10 | 2115 | pT7A3-iFLP, KJE8 |
9.76 / 52.8 |
N-His |
Tuner(DE3) |
15 ml, Rm, 0.3M IPTG RM induce o/n |
|
| 11 | 2106 | pET28b-iFLP, pLysN |
9.76 / 52.8 |
N-His |
Nico21(DE3), pLysN |
15 ml, Rm, 0.3M IPTG RM induce o/n |
|
| 12 | 2115 | pT7A3-iFLP, pLysN |
9.76 / 52.8 |
N-His |
Nico21(DE3), pLysN |
15 ml, Rm, 0.3M IPTG RM induce o/n |
|
9/12/19
2/16/19
Plasmid: 2115 pT7A3
Medium: LB + 1% glycerol + K, 15 ml
Cells: BL21(DE3) Star or Nico21(DE3)
Inducer: 1 mM IPTG
Induction, 37 oC, 30 oC, or 25 oC O/N
Lysis Buffer: 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% NP-40,, 10 mM imidazole
Inclusion Solubilization buffer: 8 M Urea, 0.1% SDS
Wash buffer: 50 mM NaPO4, pH 7.0, 1 M NaCl, 10 mM B-ME, 4 M Urea, 1% SDS, 10 mM Imidazole, 10% glycerol.
Beads: Beaver Mag Ni IDA beads, 20 ul
Protein Ladder: PageRuler Plus 250, 130, 100 70(Red), 55, 35, 25 (Red), 15, 10
Flp: 52.8 kDa
2/28/19
2/28/20
Flp 2115, transform into Bl21(DE3) Star, grow in LB+ 1% glycerol, induced with 1mM IPTG at 37 oC, 30 oC, 25 oC o/n
| Lane | Sample | |||
| 1 | Cre, 176-375 | |||
| 2 | Cre, 175-375 | |||
| 3 | 2155 Flp, from Ni column--> 1 ml SP-FF+ QFF, elute with 1.5 M NaCl | |||
| 4 | 2155 flp, purified using magnetic beads, elute with Buffer B | |||
| 5 | 2155 flp, purified from Ni column | |||
| 6 | 2155 flp, purified from Ni column, 37 oC induction, 1 L |
|||
| 7 | 2155 flp, purified from Ni column, 30 oC induction, 500 ml |
|||
| 8 | 2155 flp, purified from Ni column |
|||
| 9 | 2155 flp, purified from Ni column |
|||
| M | pageruler plus | |||