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.

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Date: 12/13/2011--12/14/2011

Page: 002870-71

Taq enzyme (377 protein) kept purifing by HiLoad^TM 16/60 Superdex^TM 200

After this, concerntrate Taq and dilyze with buffer.

Lastly, need to measure the activity by PCR. 

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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. 

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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.

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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 .

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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.

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Date:01-13-2012 through 01-17-2012;

Page:003493-94

Line 1: Positive control;

Line 2: Taq eluted with I₂₀;

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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