https://eln.excellgen.com/index.php?action=history&feed=atom&title=253_pET28b-T4_Ligase253 pET28b-T4 Ligase - Revision history2026-10-18T08:05:48ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=253_pET28b-T4_Ligase&diff=1517&oldid=prevHylei: Created page with "== '''gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp''' == '''Purpose:''' gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp. '''Date: ..."2011-08-15T18:31:32Z<p>Created page with "== '''gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp''' == '''Purpose:''' gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp. '''Date: ..."</p>
<p><b>New page</b></p><div>== '''gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp''' ==<br />
<br />
'''Purpose:''' gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp. <br />
<br />
'''Date: '''2011-8-15 <br />
<br />
'''Notebook:''' Haiyan<br />
<br />
'''Page:'''003023<br />
<br />
'''Clone ID:''' 253<br />
<br />
'''Experimenters:''' Haiyan<br />
<br />
== '''Results''' ==<br />
<br />
=== Subcloning ===<br />
<br />
Date: 2011-8-15 <br />
<br />
[[Image:IMG_1271.JPG]]<br />
<br />
PCR amplify T4 dna ligase and Digest with NdeI + xhoi, lane 1-3 <br> <br />
<br />
Digest pet 28b (lane 4) and pdbhis.mbp (lane 5, 6) with NdeI + xhoi <br> <br />
<br />
Result: T4 dna ligase no amplify bands, using fusion enzyme to amplify again. <br> <br />
<br />
=== Colony Screening ===<br />
<br />
Date: 2011-8-15<br> <br />
<br />
Make miniprep and digest with NdeI + BamH1<br> <br />
<br />
[[Image:PLentiGP-PKAalpha 6 14 2011 2.JPG]] <br />
<br />
Lane 1: pLVGP-PKAalpha 3+4, Xho1 + Mlu1 <br> <br />
<br />
Lane 2: pLentiGP #1, Xho1 + Mlu1<br> <br />
<br />
Lane 3: pLentiGP #6, Xho1 + Mlu1<br> <br />
<br />
Lane 4: pLVGP #1, Xho1 + Mlu1<br> <br />
<br />
Lane 5: pLVGP-PKAalpha 3+4, Nde1+ BamH1<br> <br />
<br />
Lane 6: pLentiGP 1, Nde1+ BamH1 <br />
<br />
Lane 7: pLentiGP 6, Nde1+ BamH1 <br />
<br />
Lane 9: pLVGP1, Nde1+BamH1 <br />
<br />
== '''Result intepretation/Summary''' ==<br />
<br />
*PKAalpha 3+4 has one internal Xho1 site. GFP-Puro can be cut out with Xho1 + Mlu1. So Lane 1 has correct number of bands. <br />
*PKA alpha 3+4 with CMV promoter can be cut out with Nde1+ BamH1. Lane 5 has correct number of bands. <br />
*lane 6 maybe good (incomplete digestion) <br />
*Sequencing Result:<br><br />
<br />
== '''Conclusion''' ==<br />
<br />
Restriction digestion <br> <br />
<br />
shows&nbsp; pLentiGP clone #1 may be good, <br> <br />
<br />
pLVGP-PKAalpha 3+4 is good<br> <br />
<br />
=== ===<br />
<br />
[[User:Paul.xu|Paul.xu]] 08:23, 15 June 2011 (EST)<br> <br />
<br />
[[Category:Cloning]]</div>Hylei