Editing PLVGP-PKA-Alpha

CloneID: 238, 239 

Date: 2011-6-9 

<br> 

Subclone PKA-alpha from pLVX-Plus2 to pLVGP<br> 

Digest pLVGP-1, pLVGP-4 with NdeI +Bamh1<br> 

Digest PKA-alpha (1+2)-pLVX-plus2 or&nbsp; PKA-alpha (3+4)-pLVX-plus2 with NdeI-BamH1<br> 

Lane 1. pLVGP-1 with NdeI +Bamh1<br> 

Lane 2 .&nbsp; pLVGP-4 with NdeI +Bamh1<br> 

Lane 3. PKA-alpha (3+4)-pLVX-plus2, NdeI-BamH1<br> 

Lane 4. PKA-alpha (1+2)-pLVX-plus2, NdeI-BamH1<br> 

<br> 

<br> 

[[Image:PLentiGP-PKA-alpha-pLVGP 6 7 2011.JPG]] 

<br> 

Ligation: <br> 

insert from lane 4 + vector from lane 1 (PKA-alpha (1+2) + pLVGP-1)<br> 

insert from lane 3 + vector from lane 2 (PKA-alpha (3+4) + pLVGP-4) 

<br> Date: 2011-6-10 Restriction digestion: 

Nde1 + BamH1<br><br> 

<br> --[[User:Hylei|Hylei]] 10:22, 10 June 2011 (EST) 

Date: 2011-6-9 

<br> 

<br> Digest pET28b-TEV 1 -4 with ECORI, SALI (Lane 9-12)<br> sequence pET28b-TEV 4 (lane 12), failed 

Digest PKA-alpha-pLVGP (1+2) 1-5 (lane 13-17); PKA-alpha-pLVGP (3+4) 1 with NdeI-BamH1 (Lane 18 )<br> 

lane 9: pET28b-TEV 1 with ECORI, SALI<br> lane 10: pET28b-TEV 2 with ECORI, SALI<br> lane 11: pET28b-TEV 3 with ECORI, SALI<br> '''lane 12: pET28b-TEV 4 with ECORI, SALI<br>''' lane 13: PKA-alpha-pLVGP (1+2) 1 with NdeI-BamH1 <br> lane 14: PKA-alpha-pLVGP (1+2) 2 with NdeI-BamH1 <br> lane 15: PKA-alpha-pLVGP (1+2) 3 with NdeI-BamH1 <br> lane 16: PKA-alpha-pLVGP (1+2) 4 with NdeI-BamH1 <br> lane 17: PKA-alpha-pLVGP (1+2) 5 with NdeI-BamH1 <br> '''lane 18: PKA-alpha-pLVGP (3+4) 1 with NdeI-BamH1 <br>''' 

<br> <br> 

[[Image:0609ECORI-SALI.JPG]]<br> 

<br> 

Digest plenti-GP 4 (lane 8), pLVGP 1-4 (lane 1-4), plenti-GP 1, 2,3 (lane 5-7) with XHOI-MLUI <br> sequence pLVGP 1, 4 (lane 1, 4), failed <br> lane 8: plenti-GP 4 with XHOI-MLUI <br> '''lane 1: pLVGP 1 with XHOI-MLUI <br>''' lane 2: pLVGP 2 with XHOI-MLUI <br> lane 3: pLVGP 3 with XHOI-MLUI <br> '''lane 4: pLVGP 4 with XHOI-MLUI <br>''' lane 5: plenti-GP 1 with XHOI-MLUI <br> lane 6: plenti-GP 2 with XHOI-MLUI <br> lane 7: plenti-GP 2 with XHOI-MLUI <br> 

<br> <br> 

[[Image:0609xhoi-mlui.JPG]] 

<br> --[[User:Hylei|Hylei]] 10:22, 13 June 2011 (EST)<br>

<br>

PKAalpha1+2 digest with NdeI+BamH1 or Xho1 +Mlu1<br>

[[Image:PKA_alpha_1_2_digestion_Xho1_Mlu1_Nde1_BamH1.JPG]]

Lane 1: PKA-alpha 1+2 in pLVGP clone #4, digest with Xho1 +Mlu1<br>

Lane 2: PKA-alpha 1+2 in pLVGP clone #5, digest with Xho1 +Mlu1

Lane 3: PKA-alpha 1+2 in pLVGP clone #4, digest with Nde1+BamH1<br>

Lane 4: PKA-alpha 1+2 in pLVGP clone #5, digest with Nde1+ BamH1<br>

Clone #5 is good.

<br><br>

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[[Category:Cloning|Cloning]]