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945 pT7A30-pMBP - Revision history
2026-10-18T08:05:04Z
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Crm at 20:17, 19 April 2018
2018-04-19T20:17:49Z
<p></p>
<p><b>New page</b></p><div>'''945 PCR'''<br />
<br />
2X PCR Buffer:Platinum Supermix HiFi<br />
<br />
Template:1281<br />
<br />
Primer:945 F+ 945 R<br />
<br />
'''Results:good, cut out and did gel purfication to prepare next step.'''<br />
<br />
[[File:945PCR.JPG|RTENOTITLE]]<br />
<br />
'''Miniprep of 945pMiniT2_947pMiniT2_3131_1727_1834'''<br />
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945pMiniT2:1-4 EcoRI<br />
<br />
947pMiniT2:5-8 n EcoRI<br />
<br />
3131:9-13 BssHII+XhoI<br />
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1727:14 XbaI+NcoI<br />
<br />
1834:15 XbaI+NcoI<br />
<br />
Buffer:Fastdigest<br />
<br />
Time:30Min<br />
<br />
'''Results:945pMiniT2 and 947pMiniT2 are good, so sent them to do sequencing. 3131 is not good, 1727 has no band, 1834 has one band.'''<br />
<br />
[[File:Miniprep of 945 947 1727 1834.JPG|RTENOTITLE]]<br />
<br />
'''Subclone of 945'''<br />
<br />
1:1727 XbaI+NcoI 173&nbsp; Cutsmart<br />
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2:1834 XbaI+NcoI 173&nbsp; Cutsmart<br />
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3:945pMiniT XbaI+NcoI 1326&nbsp; Cutsmart<br />
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4:3009pMiniT BsrGI+NheI 371&nbsp; &nbsp;NEB 2<br />
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Time:30min<br />
<br />
'''Result:1727 can see band as vector, 945pminiT2 can see band as insert, cut out and combine'''<br />
<br />
[[File:Subclone of 945.JPG|RTENOTITLE]<br />
<br />
== Date:3/16/2018 ==<br />
<br />
'''Miniprep of 945_3167'''<br />
<br />
945:1-4 Xba+NcoI Cutsmart 1326<br />
<br />
3167:5-8 BsrGI+NheI NEB 2 397<br />
<br />
Time:50min<br />
<br />
[[File:Miniprepof 945 3167.JPG|RTENOTITLE]]<br />
<br />
'''PCR Screening of 945'''<br />
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Lane 1,2: pT7A3-seq-F;945R<br />
<br />
Lane 3: 945 F; 947R<br />
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extension time:1.5min<br />
<br />
2X PCR Mix:Platinum<br />
<br />
length:1300 bp<br />
<br />
[[File:IMG 3631.JPG|RTENOTITLE]]<br />
<br />
Date:2/26/2018<br />
<br />
'''Test of 947_945_3166_3165'''<br />
<br />
{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"<br />
|-<br />
| Name<br />
| Enzymes<br />
| Size (bp)<br />
| Lane<br />
|-<br />
| 947<br />
| BglII<br />
| 646<br />
| 1,2,3<br />
|-<br />
| <br/><br />
| BssHII<br />
| 1856<br />
| 4,5,6<br />
|-<br />
| 945<br />
| BglII<br />
| 628<br />
| 11,12<br />
|-<br />
| <br/><br />
| BssHII<br />
| 1838<br />
| 13<br />
|-<br />
| 3165<br />
| SalI+BsrGI<br />
| 693<br />
| 8<br />
|-<br />
| <br/><br />
| HindIII+BsrGI<br />
| 441<br />
| 10<br />
|-<br />
| 3166<br />
| SalI+BsrGI<br />
| 1234<br />
| 9<br />
|-<br />
| <br/><br />
| XbaI<br />
| 2049<br />
| 7<br />
|-<br />
| <br/><br />
| BglII<br />
| 3259<br />
| to do<br />
|}<br />
<br />
[[File:IMG 36521.JPG|RTENOTITLE]]<br />
<br />
'''945 subclone'''<br />
<br />
'''Size:1326 bp'''<br />
<br />
'''Enzymes:Xba1+Nco1'''<br />
<br />
[[File:945subclone.JPG|RTENOTITLE]]<br />
<br />
'''Miniprep digestion of 945_986_944_3127'''<br />
<br />
{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"<br />
|-<br />
| #<br />
| Lane<br />
| Size<br />
| Enzymes<br />
| Note<br />
|-<br />
| 945<br />
| 1-4<br />
| 1326bp<br />
| Xba1-Nco1<br/><br />
| subclone<br />
|-<br />
| 986<br />
| 5-8<br />
| 774bp<br />
| HindIII+XhoI<br />
| ~4kbp<br />
|-<br />
| 944<br />
| 9-12<br />
| 2334bp<br />
| NdeI+XhoI<br />
| PCR<br />
|-<br />
| 3127<br />
| 13-16<br />
| 402bp<br />
| Nde1-HindIII<br/><br />
| Gibson Mix<br />
|}<br />
<br />
<br />
<br />
[[File:IMG 0658.JPG|RTENOTITLE]]<br />
<br />
[[Category:Cloning]]</div>
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