Guelph/9 October 2008

Today, we're lysing cells in Dave's lab.

Following the Bio-Rad Quantuum Prep recipe and soup mixes. I'm going to lyse seventeen liquid suspended colonies. Each tube came from a colony on a plate. The following is an inventory:

* crtI + crtY in pSB1A2 [#1 to #4] * crtI in pSB1A2 [#1 and #2] * crtY in pSB1A2 [#1 and #2] * crtB in pSB1A2 [#1] * frd in pSB1A2 [#B to #C] * GFP [#1 to #2]

The CRT series of genes are links in the enzymatic pipeline for β-carotene. The first entry has two carotene enzymes on one plasmid while the remaining do not. GFP is related to the RNAi project. The present bunch of cultures has been created to conform to biobrick standards so that our sequences are enclosed in the biobrick restriction sites-- this is good because it makes our sequences valid for the competition!

Centrifugation Protocol calls for 2mL tubes to be spun down-- modification: spin twice due to amount of culture fluid.

Redisolving The supernatant is discarded and the pellet is redisolved in 200uL of resuspension fluid. I used a vortex agitator to resuspend at ten to twenty seconds per tube. The time difference was based on visual inspection of the tube after beating.

Lysis 250uL lysis solution; invert 10 times, no vortex-- modification: wait 3 minutes afterward.

Neutralization 250uL neutralization solution; invert 10 times, no vortex. Centrifuge 5 minutes. Note-- centrifuge temperature was four degrees celcius, but that shouldn't affect anything. I've set the centrifuge to warm up to twenty-five, but I don't know if will reach room temperature by the end of the spin.

Collecting the plasmids... Insert 2mL wash tube. Transfer supernatant to spin column. Add 200uL matrix. Pipet mix-- modification: wait 1 minute afterward-- continuing normally: centrifuge 30 sec.

Mishap occurred here-- samples crtI-1, crtB-1, frd-B were damaged in the centrifuge: The spin columns fell off and broke. This occured because the spin columns were placed in the large eppendorfs as opposed to the small collection tubes. To recover, the matrix step was performed twice with the remains of the damaged samples. All other samples are normal.

Step: 500uL wash buffer added to column; spin 30 seconds-- modification: let sit one minute. Step: 500uL wash buffer added to column; spin 2 minutes;* spin 2 minutes. Step: Remove column to clean eppendorf; 100uL dH2O or TE buffer added; spin one minute.


 * Dropped spin column for sample CRTI + CRTY #3 here-- powder was intact, so we kept it.

Everything else went fine.

- Eddie Ma

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