June

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July

July 9th

Miniprep, digestion and gel of pSB1C3 plasmid

Goal : extract PSB1C3 from E.coli and digest it. Result : failed (gel didn't work)=> restart on 10th July

1) Mini prep to extract plasmids (Qiagen)

1- Resuspend the pelleted cells in 250 μL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

Note: Ensure RNase A has been added to the Resuspension Solution (as described on p.3)

2- Add 250 μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.

Note:. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5min to avoid denaturation of supercoiled plasmid DNA.

3- Add 350 μL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.

Note: It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris.The neutralized bacterial lysate should become cloudy.

4- Centrifuge for 5 min to pellet cell debris and chromosomal DNA.

5- Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.

Note. Close the bag with GeneJET Spin Columns tightly after each use!

6- Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

Note: Do not add bleach to the flow-through, see p.7 for Safety Information.

7- Add 500 μL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

8- Repeat the wash procedure (step 7) using 500 μL of the Wash Solution.

9- Discard the flow-through and centrifuge for an additional 1 min to remove residual. Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

10- Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 μL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.(HERE I USED 30 uL OF

Note: An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.

11- Discard the column and store the purified plasmid DNA at -20°C.

2) Digestion of miniprep product with EcoR1 & Pst1

3) Gel migration => failure (pb with gel)

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August

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October

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