IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-12: Difference between revisions

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==Redigest of Kai\X-P==
==Redigest of Kai\X-P==

Revision as of 18:47, 12 August 2006

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To-do

  • RFP in low-copy plasmid (transformed in Top10 and Top10F')
    • Colony PCR the J04450 + pSB4A3 colonies with BB primers to test if our ligation worked--Zsun 17:37, 12 August 2006 (EDT)
    • Inoculate liquid cultures with these colonies--Zsun 17:37, 12 August 2006 (EDT)
  • GFP dev in low-copy plasmid (from digest assay)
    • Run the redigest of pSB4A3 (pSB4A3\S-P, pSB4A3\X-P, psB4A3 undigested) on a gel, to see if the plasmid is the right length or if it has an insert Our gel boiled in the tank; we've labeled the tank armed and dangerous. We lost this DNA.
    • CIP treat the 4 gel-extracted pSB4A3\S-P from the digest assay
    • If the pSB4A3 plasmid looks alright, ligate GFP dev\X-P + psB4A3\S-P
    • Transform ligation products
  • Kai\X-P + J04500\S-P ligation
    • Run the KaiA\X-P, KaiB\X-P, and KaiC\X-P digests from yesterday on a gel Our gel boiled in the tank; we've labeled the tank armed and dangerous. We lost this DNA.
    • Redo the KaiA\X-P, KaiB\X-P, and KaiC\X-P digests
    • Run the KaiA\X-P, KaiB\X-P, and KaiC\X-P digests on a gel
    • Extract and purify KaiA\X-P, KaiB\X-P, and KaiC\X-P
    • CIP treat J04500\S-P
    • Ligate Kai\X-P + J04500\S-P
    • Transform ligation products
  • Growing up Kai--Zsun 17:37, 12 August 2006 (EDT)
    • Inoculate cultures of the transformed KaiA/B/C GeneART plasmids
  • More backbone
    • Grow up a lot of pSB4A3- and J04500- transformed cells for midiprepping

Results of Restreak of J00450+PSB4A3

Although the colonies took two days to grow, the restreak of the plates on Carb worked; we will now verify the ligation via PCR while growing liquid culture from them.

PCR ligation test of J04450+PSB4A3 + innoculation

Following Perry's Protocol which worked pretty well, in order to check the ligation we will do a PCR using the PCR Supermix from the VF2 and VR areas of the plasmid;

From the above image, we will try 7 colonies:

  • Top10 plate2 A (eg. 102)
  • Top10 plate1 A (eg. 101)
  • Top10F plate1 (eg. F1)
  • Top10F plate2 1 (eg. F2-1)
  • Top10F plate2 2 (eg. F2-2)
  • Top10F plate2 3 (eg. F2-3)
  • Top10F plate2 3 (eg. F2-4)

From Perry's Protocol, each will have:

  • 8uL PCR supermix
  • 1uL VF2 (2uM)
  • 1uL VR (2uM)

Then, get a carb plate, pick the desired colony, grow in 8mL LB+Carb. Choose another (monoclonal source), dip it into the 10uL rxn.

The runtime protocol, modified from Perry's is:

  • 95@15m
  • Loopx30
    • 95@30
    • 55@30
    • 72@1.30
  • 72@10
  • 4@forever


Redigest of Kai\X-P

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