Work with transformants from attempt #1 to complete second ligation
Chose seven colonies from the 2:1 insert:vector ligation plate (colonies #1-#7) and seven from the 5:1 ligation plate (colonies #8-#14) to analyze. Used each to inoculate a liquid culture, streak an index plate, and run a colony PCR reaction (after appropriate lysing in water).
Each PCR reaction mixture contained 2 uL of VF2 primer, 2 of VR primer, 9.8 uL of water, 4 uL of Phusion buffer, 0.6 uL of DMSO, 0.4 uL of dNTPs, and 0.2 uL of polymerase. They were run on the 'VR' thermocycler protocol
The PCR products were then run on a 1.0% agarose gel at 90 V vs. a 1 kb ladder.
Loaded ladder at the far left and samples 1 through 14 from left to right in order. Only sample 6 is even close the size range of the desired ligation product (8.3 kb) and even it is still at least a kb too small to be the right construct.
This day's labwork is also recorded on pages 85 and 86 of the hard copy lab notebook.
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