1995 pT7A3-Kfu-sso7d
Cloning of 1995 pT3A3-Kfu-sso7d
[edit]Purpose: Cloning of1995 pT3A3-Kfu-sso7d
Date: 2014-1----
Notebook:0
Page:
Clone ID:1995
Experimenters: shanyi
Results
[edit]cloning
[edit]PCR from 1949 pT3A3-Kfu-G590S-sso7d
fragment 1: Kfu-S590G-F+Kfu-HindIII-R
Fragment 2:Kfu-F1+Kfu-S590G-R
join two fragments using
Kfu-F1
cgacCATATGGCTAGCGCCattctggatactgactacattaccgaagatggaaagccggtcataCGTattttcaag
Kfu-HindIII-R
(GAGGGTTCTTGAAGCTTTGCTAA)
TTAGCAAAGCTTCAAGAACCCTC
clone fragment back into Nde1 + HindIII digested 1949 pT3A3-Kfu-G590S-sso7d using Nde1 + HindIII
Colony Screening
[edit]Date: 2013-10-23
Make miniprep, digest with NdeI+XHOI
Result intepretation/Summary
[edit]Conclusion
[edit]Protein Purification & QC
[edit]1/13/2018 
Storage buffer
20 mM Tris-HCl (pH 7.4 at 25 °C)
0.1 mM EDTA
1 mM DTT
100 mM KCl
stabilizers (maybe 100 mM sorbitol )
5 % trihalose
200 μg/mL BSA and 50% glycerol.
QC_5/18/2018
Phusion_homemade:original concentration:2.5mg/ml
Kfu:original concentration:1.25mg/ml
| Name | Lane | concentration | note | |
| Phusion, | 1 | 1250ng/ul | ||
| 2 | 625ng/ul | |||
| 3 | 500ng/ul | |||
| 4 | 250ng/ul | |||
| 5 | 100ng/ul | |||
| 6 | 50ng/ul | good | ||
| 7 | 30ng/ul | good | ||
| 8 | 15ng/ul | good | ||
| 9 | 6ng/ul | |||
| Kfu, | 1 | 625ng/ul | ||
| 2 | 500ng/ul | |||
| 3 | 250ng/ul | |||
| 4 | 100ng/ul | |||
| 5 | 50ng/ul | |||
| 6 | 30ng/ul | good | ||
| 7 | 15ng/ul | good | ||
| 8 | 6ng/ul | good | ||
| 9 | 3ng/ul |
