Paul.xu at 23:21, 28 March 2013

2013-03-28T23:21:39Z

New page

== '''Cloning of 1702-pET30b-A/WSN/33 M15/88K''' ==

'''Purpose:''' Cloning of 1702-pET30b-A/WSN/33 M15/88K

'''Date: '''2013-3-18---2013-3-20

'''Notebook:0'''

'''Page:'''

'''Clone ID:1702'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1702pcr1+2+1699pcr 3-18-2013.JPG]]

[[Image:1702 PCR 3-18-2013.JPG]] 

[[Image:1664+1692+1699+1702 clone 3-18-2013.JPG]] 

=== Colony Screening ===

Date: 2013-3-20

Make miniprep and digest with Nde1 + Xho1 

[[Image:1702 digest detect 3-20-2013.JPG]]

 

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

*&amp;amp;lt;br&amp;amp;gt;

== '''Conclusion''' ==

== Protein Purification ==

3/28/2013 

[[Image:1702 pET30b-A WSN 33 M15 88K.jpg]] 

M H H H H H H SSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGN K DPNNMDKAVKLYSKLKSEITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMLE HHHHHH 

pI/Mw: 8.47 / 20194.21 

 

All buffers contain 1~5 mM beta-ME to avoid aggregation. 

1: Transform Nico21(DE3)/pLysN with plasmid, grow 4 L in LB/Kan/Chl,  induce with 0.1 M IPTG o/n 37 oC. 

2. Capture using 20 ml Ni column, wash extensively with 20 mM Tris-HCl, pH7.5, 20 mM Imidazole, 300 mM NaCl, 1 mM beta-ME 

3. Elute with 20 mM Tris-HCl, pH7.5, 250 mM Imidazole, 300 mM NaCl, 1 mM beta-ME 

4. Dilute 5 folds with 20 mM Tris-HCl, pH7.5, 1 mM beta-ME to salt conc <=50 mM NaCl. 

5. Load to 20 ml SP Sepharose FF. Wash with 20 mM Tris-HCl, pH7.5, 50 mM NaCl, 1 mM beta-ME 

6. Elute with linear gradient of 20 mM Tris-HCl, pH7.5, 1 mM beta-ME, from 50 mM NaCl to 1 M over 250 ml. Protein eluted at ~200 mM NaCl. 

7. Conc fractions containing major peak using spin column. 

8. Load to gel filtration column BioSil TSK 250 equilibrated with 55 mM KH2PO4/K2HPO4, 0.2 M NaCl, 1 mM beta-ME, pH4.0., elute with the same buffer at 2.5 ml/min 

9. Collect 5 ml fractions. protein eluted between fraction 32~34. 

10. Combine fractions 32~34. Add 1 M  TECP to final conc 2 mM (100 ul of 1 M to 50 ml). Store at 4 oC. 

11. Use 15 ul for SDS-PAGE (marker is fermentas Page-Ruler Plus, 10, 15, 25 (red), 35,  55, 70(red), 100, 130, 250 kDa 

Yield: 3 tubes: 

1. 45 ml, 1.4 mg/ml, total 63 mg 

2: 40 ml, 0.8 mg/ml,  total 32 mg 

3: 45 ml, 0.5 mg/ml, total  22.5 mg 

total: 117 mg. 

 
--[[User:Paul.xu|Paul.xu]] 18:21, 28 March 2013 (EST)
 

[[Category:Cloning]]

1702-pET30b-A/WSN/33 M15/88K - Revision history

Paul.xu at 23:21, 28 March 2013