IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-12

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
  • Inoculate liquid cultures with these colonies
• 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
  • 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
  • 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
  • Inoculate cultures of the transformed KaiA/B/C GeneART plasmids

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 2mL LB+Carb. Choose another (monoclonal source), dip it into the 10uL rxn.

The runtime protocol, modified from Perry's is:

• 95@15m
• Loopx30
  • 95@1
  • 55@1
  • 72@1.30
• 72@10
• 4@forever