Revision as of 14:49, 10 September 2015

Wet Lab

We started the miniprep using the instructions of QIAprep Spin Miniprep Kit:

Add RNase A solution (200 ul of concentration mg/ul) to Buffer PI (20ml) and mix, store at 2-8˚C. This was stored in the fridge, 3rd from right. (We did not add the LyseBlue reagent to the Buffer PI )
Centrifuge the overnight culture (1ml in each tube) of 8200rpm for 3mins at room temperature
Pour out the LB, so that the pellet remains, then resuspend the pellet in 250 ug of Buffer solution. Then transfer to a microcentrifuge tube.
Add the 250ug of P2, mix thoroughly. Do not allow to proceed for more than 5 mins.
Add 350ug of Buffer N3 , mix immediately, invert 4-6 times.
Centrifuge for 13,000 rpm for 10mins in the centrifuge
Apply the supernatant, centrifuge for 30-60secs, discard flow through
Add 500ul pf PB buffer to wash and centrifuge for 30-60seconds. This was an optional step that was done in our process
Wash QIAprep spin column, add 750ul Buffer PE. Centrifuge for 30-60seconds. (We prepared glycerol stock of liquid culture, with equal parts of glycerol and cell culture of 500ul. This was stored in the -8˚C fridge.)
Centrifuge for 1 min to remove residual wash buffer.
Place QIAprep column in a clean eppendorf tube. Add 50ul of water ( not the Buffer EB), let it stand for 1 min, then centrifuge for 1 minute.

The DNA was stored in the fridge at 4 ˚C overnight.

Note: We used deionized water for step 10, so we are not sure if it is DNA free and RNA free. So we have to see if there is any degradation tomorrow.

We performed a Nano drop to check the quality of DNA. We pipetted 1ul of DNA into the machine.

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 <h1>Wet Lab<h1>
 <p class="subtitle"> Week 1: </p>
 <h3>15/07/2015 </h3>