Paul.xu at 16:10, 28 February 2020

2020-02-28T16:10:00Z

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== '''Cloning of 2115 pT7A3-iFLP''' ==

'''Purpose:''' Cloning of 2115 pT7A3-iFLP'''=='''

'''Date: '''2014-3----

'''Notebook:0'''

'''Page:'''

'''Clone ID:2115'''

'''Experimenters:'''  shanyi

== '''Results''' ==

=== CLONING ===

subclone from 2114 pEC-iFLP to  1860 pT7A3-yFLP using HindIII + Xho1 [[File:Digest for 2106 2115.JPG|RTENOTITLE]]

=== Colony Screening ===

Date: 2014

Make miniprep, digest with NdeI + HindIII + Xho1,there is no NdeI site in new FLP

[[File:2106 2115digest.JPG|RTENOTITLE]]

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

*

== '''Conclusion''' ==

== Protein Expression ==

[[File:Scan515.jpg|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width: 594px;"
|-
| Lane
| ID
| style="width: 134px;" | Protein Name
| style="width: 52px;" | MW, pI
| style="width: 44px;" | Tag
| style="width: 56px;" | Host
| style="width: 159px;" | Condition
| style="width: 78px;" | Expression
|-
| 1
| 927
| style="width: 134px;" | pET28b-Cid1 
| style="width: 52px;" | 42
| style="width: 44px;" | His
| style="width: 56px;" | Nico21(DE3), pLysN
| style="width: 159px;" | 15 ml, RM , 0.3M IPTG induce o/n
| style="width: 78px;" | Yes
|-
| 2
| 3097
| style="width: 134px;" | pET28b-pQbeta 
| style="width: 52px;" | 
| style="width: 44px;" | no
| style="width: 56px;" | Nico21(DE3), pLysN 
| style="width: 159px;" | 15 ml, RM , 0.3M IPTG RM induce o/n 
| style="width: 78px;" | do not know
|-
| 3
| 3098
| style="width: 134px;" | pET28b-AP205 
| style="width: 52px;" | 
| style="width: 44px;" | no
| style="width: 56px;" | Nico21(DE3), pLysN 
| style="width: 159px;" | 15 ml, RM , 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | do not know 
|-
| 4
| 3074
| style="width: 134px;" | pET28b-HA
| style="width: 52px;" | 
| style="width: 44px;" | no
| style="width: 56px;" | Nico21(DE3), pLysN 
| style="width: 159px;" | 15 ml, RM , 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | do not know 
|-
| 5
| 3105
| style="width: 134px;" | pET28b-AaLS 
| style="width: 52px;" | 22
| style="width: 44px;" | His
| style="width: 56px;" | Nico21(DE3), pLysN 
| style="width: 159px;" | 15 ml, RM , 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | Yes
|-
| 6
| M
| style="width: 134px;" rowspan="1" colspan="6" | 198, 98(Y), 62, 49, 38, 28, 17(Red), 14, 6, 3 
|-
| 7
| 2106
| style="width: 134px;" | pET28b-iFLP, KJE7
| style="width: 52px;" | 9.76 / 52.8 
| style="width: 44px;" | N-His
| style="width: 56px;" | Tuner(DE3) 
| style="width: 159px;" | 15 ml, Rm, 0.3M IPTG  RM induce o/n
| style="width: 78px;" | 
|-
| 8
| 2115
| style="width: 134px;" | pT7A3-iFLP, KJE7 
| style="width: 52px;" | 9.76 / 52.8 
| style="width: 44px;" | N-His 
| style="width: 56px;" | Tuner(DE3) 
| style="width: 159px;" | 15 ml, Rm, 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | 
|-
| 9
| 2106
| style="width: 134px;" | pET28b-iFLP, KJE8 
| style="width: 52px;" | 9.76 / 52.8 
| style="width: 44px;" | N-His 
| style="width: 56px;" | Tuner(DE3)
| style="width: 159px;" | 15 ml, Rm, 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | 
|-
| 10
| 2115
| style="width: 134px;" | pT7A3-iFLP, KJE8 
| style="width: 52px;" | 9.76 / 52.8 
| style="width: 44px;" | N-His 
| style="width: 56px;" | Tuner(DE3) 
| style="width: 159px;" | 15 ml, Rm, 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | 
|-
| 11
| 2106
| style="width: 134px;" | pET28b-iFLP,  pLysN 
| style="width: 52px;" | 9.76 / 52.8 
| style="width: 44px;" | N-His 
| style="width: 56px;" | Nico21(DE3), pLysN 
| style="width: 159px;" | 15 ml, Rm, 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | 
|-
| 12
| 2115
| style="width: 134px;" | pT7A3-iFLP,  pLysN 
| style="width: 52px;" | 9.76 / 52.8 
| style="width: 44px;" | N-His 
| style="width: 56px;" | Nico21(DE3), pLysN 
| style="width: 159px;" | 15 ml, Rm, 0.3M IPTG  RM induce o/n 
| style="width: 78px;" | 
|}

[[File:See blue .jpg|RTENOTITLE]]

9/12/19

[[File:2515 purification.jpg|RTENOTITLE]]

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

[[File:2115 Flp Bl21Star.jpg|RTENOTITLE]]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

[[File:2115 exp screening.jpg|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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
| 
| 
| 
|-
| 
| 
| 
| 
| 
|}

[[Category:Cloning]]

2115 pT7A3-iFLP - Revision history

Paul.xu at 16:10, 28 February 2020