IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/01

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Wet Work

 * Check biobricks I746001 and I746101

- Single colony PCR

We had 5 single colonies for each biobrick fro; yestrday. We are going to PCR each single colony.

Add 1μL of cells, 7 μL of SDW, 10μL of Phusion Flash master mix, 1μL of VF2 primer and 1μL of VR primer.

-Gel

Add 5μL of PCR products, 14μL of SDW and 1μL of dye.

-Result


 * Lane1 : HyperladderI
 * Lane2 : I746001 colony 1
 * Lane3 : I746001 colony 2
 * Lane4 : I746001 colony 3
 * Lane5 : I746001 colony 4
 * Lane6 : I746101 colony 1
 * Lane7 : I746101 colony 2
 * Lane8 : I746101 colony 3
 * Lane9 : I746101 colony 4



The result is good. Each colony of I746001 is about 1000kb (all the same size), for I746101, the size is good (about 2000kb), except for one single colony for which there is nothing. It could be a problem during the manipulation.

- Check colony 1

We have colony1 for each biobrick. We want to check them.

- Plasmid miniprep

- Digest with EcoRI and SpeI

- Run a gel (for cut plasmid, add 19μL of DNA and 1μL of dye; for uncut plasmids, add 2μL of DNA, 17μL of SDW and 1μL of dye; for PCR product, add 5μL of DNA, 14μL of SDW and 1μL of dye)


 * Lane1 : Hyperladder1
 * Lane2 : I746001 double digest (miniprep product)
 * Lane3 : I746101 double digest (miniprep product)
 * Lane4 : I746001 uncut (miniprep product)
 * Lane5 : I746101 uncut (miniprep product)
 * Lane6 : I746001 colony5 (PCR product)
 * Lane7 : I746101 colony5 (PCR product)



- Result : everything is ok except for biobrick I746001, we have only one band

- New double digest for biobrick I746001 (lane9), new gel : ok!



We are sure to have some good biobricks for agr (I746001 and I746101)


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