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Team:Valencia/Notebook/July
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=July 15th= | =July 15th= | ||
| - | We prepared YPD for stock. | + | - We prepared 0.5 L of YPD for stock. Four matraces of 250ml with 50 ml each and one bottle of 0.5 L with 300 ml. |
| + | YPD Concentration | ||
*Yeast extract ................ 10 g | *Yeast extract ................ 10 g | ||
*Peptone ..................... 20 g | *Peptone ..................... 20 g | ||
*Dextrosa (D-glucosa) ......... 20 g | *Dextrosa (D-glucosa) ......... 20 g | ||
*Agar ........................ 20 g | *Agar ........................ 20 g | ||
| - | *<math>H_{2}O</math> | + | *<math>H_{2}O</math> ......... c.s.p. 1 L |
| + | |||
| + | - One colony was observed in the pG1-APC dish and non in the pG1-NMG1 one. We re-planted the colony (single strip) and left it in the 37ºC stove. | ||
| + | |||
| + | - The LiAc transformation protocol for Yeasts was started | ||
| + | |||
| + | The yeast strains are going to be transformed with the pL2/GZ plasmid (GRE domain and LacZ reporter). | ||
| + | We planted tha yeasts in dishes with SD medium + Adenine, His. Also 200 mcl of Try and Uracil were added in each one of the dishes, because the selection marker of the plasmid is the Leu synthesis gene. | ||
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Contents |
July 8th
WETLab meeting
We start to put together all the thing, to begin working in the Lab!! We also made plans for the future.
We prepared the culture medium for growing our yeasts.
Social Event
We got together in Serranos Towers, to chill out a litle bit after the "hard work" in the lab.
July 9th
- We filled up the petri dishes with the yeast liquid culture medium.
- Then we planted the yeasts onto the petri dishes.
- We prepared an overnight culture at 37ºC into a stove with the bacteria having the plasmids pG1 and pLZ in order to purificate them next Monday.
July 10th
We transferred the bacterial culture from the stove (37ºC) to the fridge (4ºC).
July 11th
Spain won the World cup!!!!
July 12th
- We made a miniprep to purify the plasmids pG1 and pLZ.
But before you start any procedure, you have to read the protocol!!!!
- Then we carried out a digestion and the following electrophoresis to verify whether we had the constructions in the plasmids and to evaluate their concentration.
 Gabi and the high-tech device for run the gel!! |
 Gabi, Jose, Juan Angel and Dani's red hair doing the miniprep, almost done!! |
First good result!! Here a picture of the electrophoresis. From left to right: Marker, pLZ and pG1.
- We stored the non-digested plasmids in the refrigerator (we want to use it later on)
- We made (Jose made) a liquid culture of E. coli with the LEA gene, and puted it into the stove at 37ºC. Ale saying Jose what to do :D!!
 Ale saying Jose what to do :D! |
 and Jose putting the liquid medium in the tube |
- We recieved a filter paper soaked with a plasmid from Kausik Li containing an Aplysia prion fused with the GR domain. Then we put the paper containing the plasmid in water in order to transform and clone it in E. coli later on.
July 13th
- We carried out a miniprep to purify the pM2 plasmid containing the LEA gene.
- Then we carried out a digestion and the following electrophoresis to verify whether we had the constructions in the plasmids and to evaluate their concentration.
- We performed a transformation of plasmid DNA containing a prionic gene into E.coli following a heat shock protocol.
July 14th
- We prepared 600ml of yeast minimum culture medium (SD) for our future yeast cultures.
SD Composition:
- YNB w/o a.a. .............. 6.7 g (1.7 + 5)<math>(NH_4)_2 SO_4</math>
- Glucosa ................... 20 g
- Agar ...................... 20 g
- H_2O ...................... c.s.p 1 L
- We also elaborated five amino acid and nitrogen bases solutions (Leu, His, Trp, Adenine, Uracil) for which our competent yeast strains are metabolic mutants.
Solutions Composition:
- a.a. / b.n. ............... 100 mg
- H_2O d.d. ................ 50 ml
We will use them in order to differentiate between plasmid transformed and non transformed yeast colonies (Only those yeast cells carrying the plasmid with the metabolic genes that our auxotrophic mutants lack will grow).
- No bacterial growth was observed on the dishes we planted yesterday. Therefore we re-cultured the two plasmid transformed E.coli strains (with the Aplysia and yeast prion genes) we worked on July 12th with.
- We started a culture of SC 5523 and SC 2606 yeasts strains (PSI+ and PSI-) in 5 ml of YPD. Overnight incubation at 30ºC in Emilia's Lab.
July 15th
- We prepared 0.5 L of YPD for stock. Four matraces of 250ml with 50 ml each and one bottle of 0.5 L with 300 ml.
YPD Concentration
- Yeast extract ................ 10 g
- Peptone ..................... 20 g
- Dextrosa (D-glucosa) ......... 20 g
- Agar ........................ 20 g
- <math>H_{2}O</math> ......... c.s.p. 1 L
- One colony was observed in the pG1-APC dish and non in the pG1-NMG1 one. We re-planted the colony (single strip) and left it in the 37ºC stove.
- The LiAc transformation protocol for Yeasts was started
The yeast strains are going to be transformed with the pL2/GZ plasmid (GRE domain and LacZ reporter). We planted tha yeasts in dishes with SD medium + Adenine, His. Also 200 mcl of Try and Uracil were added in each one of the dishes, because the selection marker of the plasmid is the Leu synthesis gene.
