Team:UANL Mty-Mexico/Notebook

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Notebook

Reference Data

Part Name Part Short name In pSB1A3 (bp) In pSB1C3 (bp) In PUC57 (bp) Part size (bp)
RBS + TetR TetR 2925 2802 3478 732
pLac + GFP GFP 3162 3034 3715 964
pCons LacI LacI 3483 3352 4036 1,282
pTetR + mCherry mCherry 3107 2957 3660 887
BBa_K1140005 3-E1 2811 --- --- 935

This table works as a reference of the parts that were used in the laboratory and in the diary. We have a short name for each part so for example, if a diary entry says "TetR" we mean the complete part "RBS + TetR". Another important aspect is that we were working with the plasmids pSB1C3, pSB1A3 and PUC57 so here there is the information about the length of the part and the length in each plasmid.


August 7th, 2013

This day we did Minipreparation of DNA. The parts that were obtained were the following: pCos R Lac1, pLac R GFP, pTetR R mCherry, pTetR, 3-1E


August 8th, 2013

Test of mCherry with Thermo-mixer-Cualitative experiment

20 Colonies of the dish with pTet-R-mCherry were planted in 0.5 mL of Agar with Kanamycin in order to observe the red color after several hours at 42ᵒ C

Start End Hours
37ºC 2:50 pm 7:02 pm 4 hr 12 min
42ºC 7:05 pm 10:35 am 15 hr 40 min

Most tubes shown development seeing them on the light.


August 10th, 2013

20 tubes with mCherry were put in the shaker with 2 controls with CDS (with oxygen everyone).

Temperature: 37ᵒ

Revolutions: 900 rpm

Start: 11:35 am -> 9:00 am (Time 21: 35 hours)

The tubes show mCherry expression, some more intense than others.The experiment was repeated at 30°C OVERNIGHT with a volume of 500ul at 30°C with tubes of 1.5ml. They were left at 4°C meanwhile they were incubated.

Set Temperature Velocity Time Live
Black Set 42ºC 900rpm 15 hours :(
Blue Set 37ºC 900rpm 21 hours :)
Red Set 32ºC 900rpm :)

Time 10:25 – 9:15 = 22:50 hrs


August 12th, 2013

The experiment was repeated at 42°C with oxygen. It began at 2:15 pm and ended at 1:20 pm

Image 1. Tubes with the mCherry pellet that were incubated at 42ºC

August 16th, 2013

Electrophoresis gel of the digestions from 15/08/13

DNA 1x 5x
DNA 2uL
EcoRI 0.3uL 1.5uL
PstI 0.3uL 1.5uL
Buffer O 1uL 5uL
10uL 50uL

August 18th, 2013

Ligation with a 1:1 ratio DNA:Vector

Size (bp) Vector DNA:Vector
pTetR 768 2.8 0.33/1
pConsLac1 1326 1.6 0.62/1
pTetRmCherry 960 2.2 0.45/1
pLac1GFP 1005 2.1 0.47/1

This ligation was not sucessful in the transformation.


August 19th, 2013

Dishes of:PTetmCherry, PConsLac, TetR, PLacGFP, 3-1E were planted again.


August 26th, 2013

All the dishes were succesfully transformed but only the PLacGFP grew in the test tubes with medium and was succesful in the MiniPrep.


August 29th, 2013

All the dishes were succesfully transformed, we got colonies of all the parts, they grew in the test tubes and the miniPrep was sucessfully done for all.


August 30th, 2013

Digestions with EcorI. Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. *They were planted with Ampicilin.


August 31th, 2013

MiniPrep and digestions from the ones of 08/30/2013. We didn't obtained any construction.

Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP

*They were planted with Ampicilin.


September 2nd, 2013

Today we inoculated colonies for TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. We alsos prepared a digestion with EcoRI and PstI.


September 3rd, 2013

Digestion of the colonies from 09/02/2013. The ones from the gel (see image 2) were saved. The others are in a green box. We also planted mCherry and pBB1A3 vector.


September 9th, 2013

14 colonies were inoculated, 6 grew up. We are going to do new ligations.


Stability

Digestion of the colonies from 09/02/2013. The ones from the gel (see image 2) were saved. The others are in a green box. We also planted mCherry and pBB1A3 vector.


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