Monday 8 August
Purification of the digestions with ethanol before ligations has proven to be important, but we still don't get the protocol right. Give us some time to optimize it!
However some good news, the second PCR has been a complete success. We have to purify the DNA from the gel - a new protocol should be exciting, but it's becoming more like a nightmare!
Purification of DNA from agarose gel
GenElute Gel Extraction Kit from SIGMA
1.Excise band fron the agarose gel and weigh the gel
2.For every 100 mg of agarose gel, add 300 μl of Gel Solubilization Solution. Incubate the gel mixture at 55ºC for 10 minutes, or until the gel slice is completely dissolved. Vortex every 2-3 minutes.
3.Prepare binding column. Place the GenElute Binding Column G into one of 2 ml collection tubes. Add 500 μl of the Column Preparation Solution to each binding column. Centrifuge for 1 minute.
4.Add 1 gel volume of 100% isopropanol and mix until homogenous.
5.Load the solubilized gel solution mixture into the binding column. Centrifuge 1 min. Discard the flow-through
6.Add 700 μl of Wash Solution to the binding column. Centrifuge for 1 minute. Discard the flow-through and repeat this step to remove excess ethanol.
7.Transfer the binding column to a fresh collection tube. Add 30 μl of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 min.
Tuesday 9 August
The purifications from gel didn't go as expected, and to make matters worse the whole department of Genetics is in a complete chaos; there's no floor, all the important machines are hidden, and we don't have access to most of the rooms. It seems it won't get any better during the week...
Wednesday 10 August
A freshly cemented corridor separates us from the door behind which our PCR reactions are waiting for us to rescue them. As well as this, getting to the NanoDrop or the Transiluminator rooms has become an adventure due to the piles of rubble and the hanging wires we have to dodge. This department has become a modern jungle, so we try not to come over here, and stay in our downstairs lab working on more digestiones and electrophoresis.
Thursday 11 August
The department of Genetics is hardly accessible at all today, but things aren't any better here in the lab: our fridge doesn't work properly and now we have all our chemicals and DNA samples surrounded by blocks of ice.
How to fix a freezer, lesson one: gather some mops, ice trays to keep things cool and something to crush the blocks until they come unstuck. The rest is easy: open the freezer door, put your chemicals in the trays, and then charge, charge, charge at the blocks and let the ice melt. Then clean up all the mess and voila, you'll have plenty of space for all your digestions, biobricks, etc.
Friday 12 August
We finally got some ligations right, but they're too few and we're not sure whether they're OK. We'll have to study them carefully...