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Taq Purification-pET30b-sso7d-Taq d289 - Revision history
2026-10-18T02:11:35Z
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Ruili.su at 23:12, 26 January 2012
2012-01-26T23:12:56Z
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<p><b>New page</b></p><div>[[Image:Protein 377 and Taq-MutS.jpg|259x214px|Protein 377 and Taq-MutS.jpg]]<br> <br />
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Date: 12/01/11---12/06/11<br> <br />
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Notebook: Ruili Su 002864----002868<br> <br />
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*377 protein: pET30b-sso7d-Taq-d289; the size between 70 KD and 55 KD; <br><br />
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*Taq-Muts size is 128KD or so.<br />
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Line1: Taq-Muts flow through from Ni column; (10ul)<br> <br />
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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> <br />
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Line 7---10: 377 protein eluted with washing buffer (50 mM Imidazole); (10ul)&nbsp; <br> <br />
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Line 11: Taq enzyme from other. (5ul)<br> <br />
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'''Result''' <br />
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377 protein has correct size (line 7---10); also 50 mM Imidazole can comepletely elute the protein. <br />
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Taq-Muts has high level expression, it needs to pass through the Amylose beads to remove other bands. <br />
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[[Image:Taq 377-12-14-2011.jpg|281x217px|Taq 377-12-14-2011.jpg]] <br />
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Date: 12/13/2011--12/14/2011<br> <br />
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Page: 002870-71<br> <br />
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Taq enzyme (377 protein) kept purifing by HiLoad<sup>TM</sup> 16/60 Superdex<sup>TM</sup> 200 <br />
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After this, concerntrate Taq and dilyze with buffer. <br />
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Lastly, need to measure the activity by PCR.&nbsp; <br> <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; <br />
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Date: 12-22-2011<br> <br />
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Page: 002875<br> <br />
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run PCR to check the activity of Taq.<br> <br />
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1. 2mg/ml, store at 4 oC (1ul);<br> <br />
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2. 2mg/ml, store at -80 oC (1ul); <br />
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3. 4mg/ml, store at -80 oC (1ul);<br> <br />
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4. 2U/ul, control from product of Biodev, store at -20 oC (0.5ul).<br> <br />
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'''Result '''<br> <br />
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Line 1 and line 3 could happen&nbsp;? <br />
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By comparing line2 with line 4, 2mg/ml Taq should have 2u/ul activity.&nbsp; <br />
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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> <br />
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Date: 01-07-2012 through 01-12-2011<br> <br />
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Note Book: Ruili Su 2<br> <br />
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Page:003490-003492<br> <br />
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Run PCR to check Taq activity.<br> <br />
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Line1&nbsp;: Taq washed from I<sub>20</sub>&nbsp;;<br> <br />
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Line2: Taq washed from I<sub>300</sub><br> <br />
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Line3: Taq washed from I<sub>50</sub>&nbsp;;<br> <br />
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Line4: Taq from supertanant.<br> <br />
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[[Image:Taq 377-01-17-2012.jpg|162x267px|Taq 377-01-17-2012.jpg]] <br />
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Date: 01-07-2012 through 01-13-2011 <br />
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Note Book: Ruili Su 2 <br />
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Page:003490-003493<br> <br />
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Run SDS-PAGE to check the purity of Taq. <br />
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Line1: supertanant; <br />
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Line2: Taq washed from I<sub>20</sub>&nbsp;; <br />
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Line3: Taq washed from I<sub>50</sub>&nbsp;; <br />
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Line4: Taq washed from I<sub>300</sub> . <br />
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'''Result''' <br />
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Taq from I<sub>20</sub> and I<sub>50</sub> have activity,and supertanant also. <br />
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Taq from I<sub>20</sub> has high purity and quantity.<sub></sub> <br />
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So, collect the Taq from I<sub>20</sub> to do concerntration. <br />
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[[Image:Taq 377 PCR for activity 1-12-2012-1.JPG]] <br />
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Date:01-13-2012 through 01-17-2012; <br />
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Page:003493-94 <br />
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Line 1: Positive control; <br />
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Line 2: Taq eluted with I<sub>20</sub>; <br />
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Date:01-24-2012 <br />
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[[Image:Taq 377 PCR --measure Unit-1-25-2012.JPG|242x217px|Taq 377 PCR --measure Unit-1-25-2012.JPG]] <br />
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Experimenter: Yingying Lu <br />
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<br> Line1: Taq after concentration: <br />
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Line2: 1:2 Taq diluted with storage buffer; <br />
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Line3: 1:5 <br />
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Line4: 1:10 <br />
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Line5: 5U/ul<br> <br />
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Line6: NEB One Taq 5U/ul <br />
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Line7: SF Taq 2U/ul <br />
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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<br />
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Page:003501<br />
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Experimenter: Ruili Su<br />
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Line1: 1:5 diluted taq with storage buffer;<br> <br />
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Line2: 1:25 diluted taq <br />
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Line3: 1:5<sup>3</sup><sup></sup> diluted taq <br />
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Line4: 1:5<sup>4 </sup> diluted taq <br />
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Line5: 1:5<sup>5 </sup><sup></sup>diluted taq <br />
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Line6: 1:100 diluted taq <br />
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Line7: Invitrogen Taq 5U/ul <br />
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Line8: NEB One Taq 5U/ul <br />
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[[Category:Protein_Purification]]</div>
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