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Team:WashU/Notebook/Yeast
From 2010.igem.org
Week of 6/21
6/24
plated Yeast Gal-YFP MATa, trp1, leu2::GAL1-YFP URA3/Kanr and Yeast YF4 MATa Prototroph on a Kan plate and a YPD plate
Started a liquid culture of Yeast YF4 MATa Prototroph in YPD
Week of 6/28
6/29
A practice transformation was preformed at the Cohen Lab with Ilaria. The OD600 was 0.651 which was low, but used in order to speed up the experiment. The PMKV005 loxKanlox Plasmid (McCusker 2005 Yeast, 21: 163-171) was used into transform wildtype yeast. The plates used were Kan positive plates created on 6/24. However it was later found that these plates might not correctly select for drug resistance. Two plates were made, one with the transformant and one with a control. The attached procedure was used and annotated during the experiment
7/1
• Examined the Sample and Control plates from the practice transformation on June 29th. Two colonies had grown on the Sample plate and none showed on the control plate. The low colony number could be due to a low OD600 (0.651) used in the transformation. • Plated the two colonies on a G418 plate in order to conduct the first round of purification
7/2
PRACTICE YEAST TRANSFORMATION Colonies grew on the first purification plate for both colony 1 and 2. Two colonies were plated from both transformed colony 1 and transformed colony 2 onto a second and final purification plate -- PLATES: YPD: all yeast grew His-: only 1&2 (WT) yeast grew, (4,6,11,12 did not grow) Kan:3,4,7 grew- 1&2 were selected against but some colonies were visible Ura-: only 1&2 (WT) yeast grew, (11 did not grow)
Amp: bacteria 8,9,10 grew (other amp plate WT control did not grow)
conclusion: all the resistance plates work in that they select against those w/o the resistance; however, there was some low level of growth on some of these plates where there was not supposed to be
Week of 7/12
7/12
streaked out wild type yeast strain FY4 onto a YPD plate and inoculated about 5 mL of YPD- incubate and shake Brian: The FY5 freezer stock was accidentally allowed to thaw and therefor a second freezer stock was started by growing an overnight culture inoculated from the original freezer stock. The freezer stock was made according to protocol from the cohen lab.
7/13
Plated FY4 yeast onto a YPD plate to grow up for a practice transformation in case the plate from the freezer stock does not grown since there were no visible growths on it. The FY4 was taken from a Ura- plate plated on 6/29 because it had the best colonies out of all the plates in the refrigerator. A freezer stock of FY4 was made according to protocol from an overnight culture of the FY4 strain since the original stock was destroyed when plating on 7/12.
7/14
Yeast colonies were observed on the FY4 plated on 7/12 from the FY4 freezer stock. This plate was brought to the Cohen lab along with liquid YPD-4 and they were used to inoculate a 5ml overnight culture.
7/15
Yeast Transformation Alice and Brian went to the Cohen Lab to preform a yeast transformation. Ilaria showed us how to use the nano-drop using the following procedure:
1. Wipe the nanodrop with a kim-wipe 2. Click the shortcut on the computer that is identicle to the logo on the nano drop 3. Choose Nucleic Acid in the program 4. Put on 1 μL of dH2O to clean it 5. Cleantop and bottem with kim wipe 6. Put on 1 μL of blank 7. Clean with Kim wipe 8. Put on 1 μL of plasmid 9. Make sure sample type is set to DNA 50 10. Look at the graph, a spike at 130 is due to salts, a spike at 260 is due to DNA, a spike at 280 is due to protein. Record the 260/230 ratio and 260/280 ratios, you want then both to be above 1, somewhere between 1-2 is good
A DNA concentration of ~1ng/ μL was measured which is extremely low, and therefor the plasmid should not be used to preform a yeast transformation. This could be a poissible explination for the extremely low transformation efficiency of Transformation-1. Because of this it is necessary to amplify the plasmid in E. coli. Amplification of PMKV005 The PMKV005 was to be amplified in E. coli to increase its concentration. Vorachek-Warren et. al. 2004 showed that PMKV005 was derived from the pAG26 plasmid which had ampicillin resistance. The competent cells were obtained from NEB thawed on ice and transformed with the following protocol.
1. Pipette 1 μL of plasmid into the cells. Flick the tubes very gently; 2. Let them site on ice for 10 minutes; 3. heat shock at 42 degC for 45 seconds; 4. Return tubes on ice; 5. add 250 μL SOC media (LB works too but SC was used); 6. grow cells at 37 degC 225 rpm for 1 hour; Placed in incubator directly to the left of the shaker in the cohen lab 7. Plate 50 μL on a antibiotic resistant plate (which you have warmed up with the lid slightly open for about 20 minutes); 8. incubate overnight at 37 degC.
7/16
Plated a colony from the PMKV005 e. coli transformation on 7/15 into LB for an overnight culture.
7/17
Conducted a Miniprep from the o/n culture of PMKV005 transformed E. coli made on 7/16. Conducted using the Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge protcol on page 22 of the attached manual. All places when centrifugation is between 30-60 seconds 60 seconds were used.
The 3 LB plates from the Mol. Bio. group were also examined and no colonies were observed.
