Taq Purification-pET30b-sso7d-Taq d289
Date: 12/01/11---12/06/11
Notebook: Ruili Su 002864----002868
- 377 protein: pET30b-sso7d-Taq-d289; the size between 70 KD and 55 KD;
- Taq-Muts size is 128KD or so.
Line1: Taq-Muts flow through from Ni column; (10ul)
Line 2: Taq-Muts washed with buffer A (10 mM Imidazole); (10ul)
Line 3---6:Taq-Muts eluted with buffer B (300 mM Imidazole); (10ul)
Line 7---10: 377 protein eluted with washing buffer (50 mM Imidazole); (10ul)
Line 11: Taq enzyme from other. (5ul)
Result
377 protein has correct size (line 7---10); also 50 mM Imidazole can comepletely elute the protein.
Taq-Muts has high level expression, it needs to pass through the Amylose beads to remove other bands.
Date: 12/13/2011--12/14/2011
Page: 002870-71
Taq enzyme (377 protein) kept purifing by HiLoadTM 16/60 SuperdexTM 200
After this, concerntrate Taq and dilyze with buffer.
Lastly, need to measure the activity by PCR.
Date: 12-22-2011
Page: 002875
run PCR to check the activity of Taq.
1. 2mg/ml, store at 4 oC (1ul);
2. 2mg/ml, store at -80 oC (1ul);
3. 4mg/ml, store at -80 oC (1ul);
4. 2U/ul, control from product of Biodev, store at -20 oC (0.5ul).
Result
Line 1 and line 3 could happen ?
By comparing line2 with line 4, 2mg/ml Taq should have 2u/ul activity.
Date: 01-07-2012 through 01-12-2011
Note Book: Ruili Su 2
Page:003490-003492
Run PCR to check Taq activity.
Line1 : Taq washed from I20 ;
Line2: Taq washed from I300
Line3: Taq washed from I50 ;
Line4: Taq from supertanant.
Date: 01-07-2012 through 01-13-2011
Note Book: Ruili Su 2
Page:003490-003493
Run SDS-PAGE to check the purity of Taq.
Line1: supertanant;
Line2: Taq washed from I20 ;
Line3: Taq washed from I50 ;
Line4: Taq washed from I300 .
Result
Taq from I20 and I50 have activity,and supertanant also.
Taq from I20 has high purity and quantity.
So, collect the Taq from I20 to do concerntration.
Date:01-13-2012 through 01-17-2012;
Page:003493-94
Line 1: Positive control;
Line 2: Taq eluted with I20;
Date:01-24-2012
Experimenter: Yingying Lu
Line1: Taq after concentration:
Line2: 1:2 Taq diluted with storage buffer;
Line3: 1:5
Line4: 1:10
Line5: 5U/ul
Line6: NEB One Taq 5U/ul
Line7: SF Taq 2U/ul
Notebook: Ruili Su 2
Page:003501
Experimenter: Ruili Su
Line1: 1:5 diluted taq with storage buffer;
Line2: 1:25 diluted taq
Line3: 1:53 diluted taq
Line4: 1:54 diluted taq
Line5: 1:55 diluted taq
Line6: 1:100 diluted taq
Line7: Invitrogen Taq 5U/ul
Line8: NEB One Taq 5U/ul