IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-12: Difference between revisions
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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. | 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== | ==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; | 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; | ||
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*1uL VR (2uM) | *1uL VR (2uM) | ||
Then, get a carb plate, pick the desired colony, | 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: | The runtime protocol, modified from Perry's is: | ||
Revision as of 13:07, 12 August 2006
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)
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
