S-STEM Scholar Kieran Younger's Blog

Cell Lysing  Over the past few weeks, I've been learning how to lyse cells with my lab partner, Alex. We started with a paper (Kikuchi) on lysing Deinococcus radiodurans - then using the information from it's protocol to try and lyse cells that are  Deinococcus aquaticus .  Here's the protocol we pulled from the paper -  MATERIALS AND METHODS D. radiodurans MR., KR, (I1) and Sark were used in this experi-ment. One milliliter of overnight culture was inoculated into 200 ml of TGY medium (1). The bacterial cells were harvested after cultivation from 24 to 36 h at 30°C. The cells were washed twice with Multibuffer (50 mM sucrose, 10 mM Tris-HCI, 40 mM EDTA, 0.1% Triton X-100, pH 8.0) and were lysed by the incubation with lysozyme (1 mg/ml) for 5 min at 37°C. Plasmids were isolated by the alkaline lysis method (12). For the analysis of plasmids, agarose gel electrophoresis was carried out using Tris-acetate buffer (12) at 100 V constant voltage. Two agarose concentration...

 Fourth Week Fun     This week, we ran a second large gel holding a different set of samples from last week. We used the separate set hoping that it would show up on the gel, as the samples the previous week didn't show on the gel at all. After running the gel, not even primers showed up, which is why we ran the second set of samples, hoping that we would get results. We added 10.75  μl   of mastermix, .5  μl  FWD, .5  μl  DNA, and 37.75  μl of PCR water. After loading the results on a gel, we let it run for about 2 hours. After running the gel again we got the same results as the previous week, however, it's very possible no primers we added to the samples. Another thing I did was learn more about enzyme restriction/ligation from Lizzy and Jonathon, and how they might change their protocol to help with the DNA's binding.  New Protocol for Enzyme Restriction/Ligation      1. Measure the DNA concentration of inser...