7:30 am: Picking up by AIMS Commute Car.
8:10 am: Got to AIMS. Switch on LapTop & checking mails.
8:30 am: Set a gel for electrophoresis to check the PCR.
8:35 am: Made Media for Cloning.
8:45 - 9:00 am: Had a look some website to search the way to solve some problem with DGGE.
9:00 - 9:30 am: Talked with David about DGGE.
- some ways I considered it might be better;
changing conc. of gel, voltage pressure, PCR primers, thermal cycle, etc...
- is it worth to try changing conc. of gel (from 50-70% to 45-65%)?
Last time of DGGE with 50-70% gel didn't work like just smear on the gel.
And, the result of that Lone and Matthias tried with 45-65% gel worked really well,
like they've got really clear bands like that they could cut them out from gel.
9:30 am: Ran PCR products on a gel to check them.
9:40 -11:30 am: Made the Plan how to go DGGE. Afterwards, made up 100 ml of 100% & 100 ml of 0% solution for DGGE. Started Extraction of Secondary Metablites from fungi in Chemistry Lab at the same time. Took membranes I prepared for Colony Hybridization yesterday out of incubator and started Cell Debris. Checked gel and took image of PCR products.
11:30 am - 13:30 pm: Started setting gel for DGGE. Made both of 45-65% of gel and 45-70% of gel to compare if they works or not. Made some LB agar plates used sterilized Media that I made this morning. Precipitation of PCR products with EtOH (just added 3M sodium acetate and ice-cold 100 EtOH and put tubes in -20℃ Freezer overnight). Kept doing Extraction of Secondary Metabolites and Cell Debris.
13:30 - 14:00 pm: Lunch. Checked some mails.
14:00 - 15:30 pm: Started Cloning for checking if New (cheaper) cloning kit works (15 min for Ligation and 1.5 hours for Transformation including incubation time). During incubation, prepared LB plate (+km, Amp and X-gal) for inoculation onto them. Incubate membranes at 42℃ with shaking overnight. Extraction of Secondary Metabolites as well. PCR for V. mimicus and V. corallolyticus for making DGGE controls (this PCR hasn't been working so well that I have to try it again and again...)
15:30 - 16:00 pm: Inoculation onto LB (+Antibiotics) agar Plates. Put them in incubator at 37℃ overnight. Added EtOAc into 2 more new fungi cultures to extract products.
16: 00 - 16:30 pm: Found some problem from works (eg. no bands on the gel, there are no X-gal stocks in Freezer, should I purify PCR products for DGGE, etc..) Finish precipitation of PCR products with EtOH. Mailed to David about some result and further process.
16:40 am: Back to home.
Agenda for next day
- Keep going Extraction of Secondary Metabolites (evapolate EtOAc)
- Wash membranes and scan one of them
- Stain DGGE gel and take images
- Make colony libraries if cloning kit worked well
- Make liquid cultures of some of above clones to check if inserts are exactly in cells
こーやって一日の仕事書くとやってる！って感じがした。
でも定時に帰れたし、そんなにきつくは無いか。
これでまた他の単離培養とか新しいキットの検討とか文献探しとか出てきたらぐるぐるするかも。
てか、何回DGGEやったらいいのやら。
機械新しくなってから(大学時代使った奴と同じタイプ)全くいい結果が出ない。
くっそーーーー！おかげでサンプル作りも何度もやらねばならないではないか！
がんばろ。Be patientだ。
できたら、もーちょっと語彙増やしたいな。
書いてて思ったけど同じ語彙＆同じ文法しか使ってない気がする。
・・・意思疎通できればいっか。