Editing Taq Purification-pET30b-sso7d-Taq d289

[[Image:Protein 377 and Taq-MutS.jpg|259x214px|Protein 377 and Taq-MutS.jpg]]<br> 

Date: 12/01/11---12/06/11<br> 

Notebook: Ruili Su 002864----002868<br> 

*377 protein: pET30b-sso7d-Taq-d289; the size between 70 KD and 55 KD; <br>

*Taq-Muts size is 128KD or so.

Line1: Taq-Muts flow through from Ni column; (10ul)<br> 

Line 2: Taq-Muts washed with buffer A (10 mM Imidazole); (10ul)<br> Line 3---6:Taq-Muts eluted with buffer B (300 mM Imidazole); (10ul)<br> 

Line 7---10: 377 protein eluted with washing buffer (50 mM Imidazole); (10ul)&nbsp; <br> 

Line 11: Taq enzyme from other. (5ul)<br> 

'''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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[[Image:Taq 377-12-14-2011.jpg|281x217px|Taq 377-12-14-2011.jpg]] 

Date: 12/13/2011--12/14/2011<br> 

Page: 002870-71<br> 

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

After this, concerntrate Taq and dilyze with buffer. 

Lastly, need to measure the activity by PCR.&nbsp; <br> 

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[[Image:Taq 377 PCR for activity 12-20-1022.JPG|215x284px|Taq 377 PCR for activity 12-20-1022.JPG]]&nbsp; 

Date: 12-22-2011<br> 

Page: 002875<br> 

run PCR to check the activity of Taq.<br> 

1. 2mg/ml, store at 4 oC (1ul);<br> 

2. 2mg/ml, store at -80 oC (1ul); 

3. 4mg/ml, store at -80 oC (1ul);<br> 

4. 2U/ul, control from product of Biodev, store at -20 oC (0.5ul).<br> 

'''Result '''<br> 

Line 1 and line 3 could happen&nbsp;? 

By comparing line2 with line 4, 2mg/ml Taq should have 2u/ul activity.&nbsp; 

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[[Image:Taq 377 PCR for activity 1-12-2012.JPG|166x254px|Taq 377 PCR for activity 1-12-2012.JPG]]<br> 

Date: 01-07-2012 through 01-12-2011<br> 

Note Book: Ruili Su 2<br> 

Page:003490-003492<br> 

Run PCR to check Taq activity.<br> 

Line1&nbsp;: Taq washed from I<sub>20</sub>&nbsp;;<br> 

Line2: Taq washed from I<sub>300</sub><br> 

Line3: Taq washed from I<sub>50</sub>&nbsp;;<br> 

Line4: Taq from supertanant.<br> 

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[[Image:Taq 377-01-17-2012.jpg|162x267px|Taq 377-01-17-2012.jpg]] 

Date: 01-07-2012 through 01-13-2011 

Note Book: Ruili Su 2 

Page:003490-003493<br> 

Run SDS-PAGE to check the purity of Taq. 

Line1: supertanant; 

Line2: Taq washed from I<sub>20</sub>&nbsp;; 

Line3: Taq washed from I<sub>50</sub>&nbsp;; 

Line4: Taq washed from I<sub>300</sub> . 

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'''Result''' 

Taq from I<sub>20</sub> and I<sub>50</sub> have activity,and supertanant also. 

Taq from I<sub>20</sub> has high purity and quantity.<sub></sub> 

So, collect the Taq from I<sub>20</sub> to do concerntration. 

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[[Image:Taq 377 PCR for activity 1-12-2012-1.JPG]] 

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

Page:003493-94 

Line 1: Positive control; 

Line 2: Taq eluted with I<sub>20</sub>; 

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Date:01-24-2012 

[[Image:Taq 377 PCR --measure Unit-1-25-2012.JPG|242x217px|Taq 377 PCR --measure Unit-1-25-2012.JPG]] 

<br> 

Experimenter: Yingying Lu 

<br> Line1: Taq after concentration: 

Line2: 1:2 Taq diluted with storage buffer; 

Line3: 1:5 

Line4: 1:10 

Line5: 5U/ul<br> 

Line6: NEB One Taq 5U/ul 

Line7: SF Taq 2U/ul 

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[[Image:Taq 377 PCR --measure Unit 2-1-25-2012.JPG|239x241px|Taq 377 PCR --measure Unit 2-1-25-2012.JPG]]<br>Notebook: Ruili Su 2

Page:003501

Experimenter: Ruili Su

Line1: 1:5 diluted taq with storage buffer;<br> 

Line2: 1:25 diluted taq 

Line3: 1:5<sup>3</sup><sup></sup> diluted taq 

Line4: 1:5<sup>4 </sup> diluted taq 

Line5: 1:5<sup>5 </sup><sup></sup>diluted taq 

Line6: 1:100 diluted taq 

Line7: Invitrogen Taq 5U/ul 

Line8: NEB One Taq 5U/ul 



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[[Category:Protein_Purification]]