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Thursday, February 24, 2005Result!!! (of a sort)I finally appear to have cracked my southern blot problem. This morning I printed the results of my latest southern blot - a whole 11 minutes before my "progress" meeting with SWMNBN.Over the past few days I have worked truly horrendous hours. Friday I worked from 10am until 11pm, on my last attempt at blotting. I then came in saturday and washed my membranes - only to find that there was no radioactivity showing on the membrane. Nevertheless I laid it down in a cassette overnight before going to a birthday party. Sunday, as expected - no result. The way a Southern blot works (continuing from Stress is making me ill), is that genomic DNA is cut into fragments and spread out along a gel. The DNA is then "blotted" - transferred to a Nylon membrane. The DNA then needs to be "probed" with a radioactive DNA fragment for visualisation. Probing relies on an intrinsic property of the DNA double helix - strands of DNA like to pair up. They do so because A always binds to T and C always binds to G. So if I have a single strand of DNA consisting of the sequence ACTGACTG, then its "complementary strand" TGACTGAC will stick to it, forming a double helix. The DNA bound to the membrane has been chemically treated so that all of the DNA is single stranded - and thus is looking for a complementary strand (my probe) to stick to. After making my probe (a short stretch of DNA identical to the gene that I am looking for that I have made radioactive), the last step is to boil it. This causes the 2 strands of the double helix to separate and become double stranded. Left to its own devices, this single stranded DNA, will form double stranded DNA again as it cools to between 50C to 60C. To stop this, the probe DNA is plunged onto ice to snap cool it as soon as it is boiled, thus remaining single stranded. The membrane from the blot is placed into a heated oven, in glass tubes with caps at either end. In to this is poured a soapy solution called hybridisation solution. The bottles rotate slowly, ensuring that the solution sloshes constantly over the surface of the membrane. To this bottle is added the radioactive DNA probe. The single stranded DNA is heated to 65C in the oven. Now single stranded DNA at 65C is ready for action! Its all loved up and desperately wants to partner up with another, complementary piece of DNA! Woohoo! Party! As the probe sloshes around the bottle it inevitably comes into contact with some of the single-stranded DNA stuck to the membrane. If it finds a piece of DNA complementary to it self is sticks. That spot on the Nylon membrane is now slightly radioactive. After about 16 hours (ie overnight), the solution in the bottle is discarded and the membrane is washed repeatedly in a slightly less soapy buffer to remove any unpaired single stranded probe molecules. Crucially, the paired probe remains bound to the DNA stuck in the membrane. Thus the membrane has patches of radioactivity on it. All previous blots have failed at this stage, with no radioactive probe bound to the membrane. After getting completely shit-faced at the birthday party on saturday night, I dragged myself delicately into the lab sunday evening. I had a little over 60 hours until my next "progress" meeting with SWMNBN and had no choice but to start again from scratch. I remade all of my solutions from scratch, adjusting them to a precise pH and digested more DNA. I finished at 3am. Monday I dragged myself into the lab after lunch. Time until meeting: 44 hours. I ran the gel, before using a new protocol to blot the gel. 8 hours later I had a nylon membrane, hopefully with DNA bound. Normally, one would wrap the membrane in clingfilm, stick it in the fridge and come back the next day. That wasn't an option, with the meeting looming. Wearily, I started to make my DNA probe, before adding it to the bottles with the membrane to hybridise overnight. I left the lab at 6am and caught the first bus of the day home to bed. At lunchtime, my alarm clock woke me so that I could write the pre-meeting progress report demanded by SWMNBN. Time until meeting: 22 hours. After snatching a few hours more sleep, I headed back into the lab to wash the excess probe off the membrane. Now things were looking promising, as I ran the geiger counter over the membrane - it started to click. Yes!!! Some radioactive probe had bound to the membrane. It wasn't much granted - the radiation level rose from 0.5 counts per minute (normal background in the lab) to 2 counts per minute (probably the same as a large lump of granite!), but I'll take it! I carefully laid the membrane flat in a lead-lined cassette and placed a radiation sensitive screen in there with it. I then placed it to one side. Time to meeting: 10 hours. After lying awake most of the night worrying, I crawled into the lab. I placed the radiation sensitive screen into the scanner and sat back. Miracle of miracles an image appeared of a the nylon membrane - complete with faint black bands where I hoped the gene I was looking for would be. YES!!!!!!! Time until meeting: 11 minutes.... The meeting went as expected. I had been prewarned that SWMNBN was furious with my lack of progress to date and "looking to tear me a new arsehole". It came to my turn to get grilled, and she turned over a copy of my "progress" report - covered in red ink and angry looking scribbles. "Before we start," I said, "I just want to show you this - hot off the laser printer" and handed her the printout. Flummoxed!! I could see that she was seething and absolutely torn. On the one hand, we had the first positive results in months - on the other hand I am sure she had spent hours in front of a mirror practising what she was going to say. After grilling me for 30 minutes with little conclusive to say the meeting concluded. "Well you've made some small progress. Now can you press the accelerator pedal a little harder and get us the data we need for the conference next month.." |
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