PLVGP-PKA-Alpha

CloneID: 238, 239

Date: 2011-6-9

Subclone PKA-alpha from pLVX-Plus2 to pLVGP

Digest pLVGP-1, pLVGP-4 with NdeI +Bamh1

Digest PKA-alpha (1+2)-pLVX-plus2 or  PKA-alpha (3+4)-pLVX-plus2 with NdeI-BamH1

Lane 1. pLVGP-1 with NdeI +Bamh1

Lane 2 .  pLVGP-4 with NdeI +Bamh1

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

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

Ligation:

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

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

Date: 2011-6-10 Restriction digestion:

Nde1 + BamH1

--Hylei 10:22, 10 June 2011 (EST)

Date: 2011-6-9

Digest pET28b-TEV 1 -4 with ECORI, SALI (Lane 9-12)
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 )

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

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

--Hylei 10:22, 13 June 2011 (EST)

PKAalpha1+2 digest with NdeI+BamH1 or Xho1 +Mlu1

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

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

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

Clone #5 is good.