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Team:UANL Mty-Mexico/Notebook
From 2013.igem.org
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
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.
