IGEM:Paris Bettencourt 2012/Notebook/RE group/Lab notes
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==21.07.2012== | ==21.07.2012== | ||
| - | === | + | ===Transformation results=== |
| - | + | Successful transformation: | |
| - | + | * [http://partsregistry.org/Part:BBa_K098991 BBa_K098991] | |
| - | + | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_K093012 BBa_K093012] | |
| - | + | * [http://partsregistry.org/Part:BBa_B0032 BBa_B0032] | |
| - | + | ||
| - | + | Unsuccessful transformation: | |
| - | + | * [http://partsregistry.org/Part:BBa_R0051 BBa_R0051] | |
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===Start Culture for miniprep and glycerol=== | ===Start Culture for miniprep and glycerol=== | ||
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<ul> | <ul> | ||
| - | <li> We pick 2 colonies from each plate.</li> | + | <li> We pick 2 colonies from each plate with successful transformation.</li> |
<li> Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).</li> | <li> Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).</li> | ||
Revision as of 12:32, 21 July 2012
Contents |
22.07.2012
Glycerol Stock
| Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name |
|---|---|---|---|---|---|
| | | | | | |
| | | ||||
| | | | | | |
| | | ||||
| | | | | | |
| | |
21.07.2012
Transformation results
Successful transformation:
Unsuccessful transformation:
Start Culture for miniprep and glycerol
- We pick 2 colonies from each plate with successful transformation.
- Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).
- Overnight incubation.
Colony PCR of I13453 and E0040
- Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently vortex the samples and spin down.
- When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Chemical | Volume |
|---|---|
| | |
| | |
| | |
| | |
| | |
| | |
| Step | Temperature, °C | Time | Number of cycles |
|---|---|---|---|
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
- Comments: LID = 100°C
20.07.2012
Biobrick Retrieval Protocol was used. List of retrieval parts:
| Part | Description | Location in a kit | Plasmid Backbone |
|---|---|---|---|
| | | | |
| | | | |
| | | | |
| | | | |
Heat Shock Transformation
- Parts from the list above were transformed using Heat Shock Transformation protocol:
- 20ml of competent NEB Turbo cells + 2ml of the plasmids.
- The remaining plasmids (8ml) were stored at -20°C.
16.07.2012
Designing primers for IsceI and pBAD+IsceI from SMR6316 cells
14.07.2012
Restriction Digestion
- The following parts were digested with EcoRI and PstI restriction enzimes:
| Part | Description | Plasmid Backbone | Size, bp | Stock name |
|---|---|---|---|---|
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | |
- The following protocol was used:
| Chemical | Volume |
|---|---|
| | |
| | |
| | |
| | |
| | |
- After 10 min at 37°C (???)
Gel
We run a gel to control colony PCR results. To do the PCR we use the next primers:
- Forward primer: VF2
- Reverse primer: VR
From left to right:
- Ladder = 5 ml;
- pRG.007 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
- pRG.008 (GFP, BBa_E0240) = 5ml + 1ml of dye = 6ml. Expected size: 720bp
- pRG.009 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.010 (pBad, BBa_K117004) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.011 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.012 (pBad, BBa_I746902) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.013 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.014 (pBad, BBa_J04450) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.015 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.016 (pBad, BBa_I13540) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- pRG.017 (pBad, BBa_R0011) = 5ml + 1ml of dye = 6ml. Expected size: 130bp
- Ladder = 5 ml;
12.07.2012
Results of sequencing
Sequencing results for PLac and PBAD were consistent.
Start Culture of E0240, K117004, I746902, J04450, I13540, I13540, Control + R0011 trasformant
- Using two colonies that formed (colonies 1 and 2) in each plate (A, B and C)
- 6 mL of LB + Ampicilline
- Incubate over night
| Part | Description | Colony | Name |
|---|---|---|---|
| | | | |
| | | ||
| | | | |
| | | ||
| | | | |
| | |
PCR of E0240, K117004, I746902, J04450, I13540, I13540, Control + R0011
- Protocol from 07.07.12
- DNA taken by picking colony from plates (nothing had grown in plate containing J09250)
| Biobrick name | Colony | Tube n° | Size of fragment |
|---|---|---|---|
| BBa_E0240 | 1 | 1 | 1076 |
| BBa_E0240 | 2 | 2 | 1076 |
| BBa_K117004 | 1 | 3 | 12286 |
| BBa_K117004 | 2 | 4 | 12286 |
| BBa_I746902 | 1 | 5 | 2311 |
| BBa_I746902 | 2 | 6 | 2311 |
| BBa_J004450 | 1 | 7 | 1269 |
| BBa_J004450 | 2 | 8 | 11269 |
| BBa_I13540 | 1 | 9 | 1269 |
| BBa_I13540 | 2 | 10 | 1269 |
| Control + (R0011) | 1 | 11 | 255 |
| Control + (R0011) | 1 | 12 | 255 |
11.07.2012
Sequencing pLac and pBAD
- We want to sequence pBAD (tubes pRG003 and pRG004) and pLac (tube pRG005)
- For sequencing,we need have 30uL (next time quantity should be 20uL) at a concentration of 50ng/uL (any concentration between 30 and 100ng/uL is good) of our plasmids in a 1.5mL eppendorf
- The primers we used are VR and VF2 at a concentration of 10uM
| Biobrick name | Name of tube in which it can be find & Concentration (ng/uL) | Name of tube we are making & Concentration (ng/uL) | Qt that needs do be taken from existing tube(uL) | Qt of DNAse free water to add (uL) |
|---|---|---|---|---|
| BBa_I13453 | pRG003 & C= 246.4 | pRG003.s & C= 30 | 6.1 | 23.9 |
| BBa_I13453 | pRG004 & C= 191.6 | pRG004.s & C= 30 | 7.8 | 22.2 |
| BBa_R0011 | pRG005 & C= 279.8 | pRG005.s & C= 30 | 5.4 | 24.6 |
- We sent our tubes to be sequenced by gatc [1]
Biobrick Retrieval
Biobrick Retrieval Protocol was used. List of retrieval parts:
| Part | Description | Location in a kit | Plasmid Backbone |
|---|---|---|---|
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
Heat Shock Transformation
- Parts from the list above were transformed using Heat Shock Transformation protocol:
- 20ml of competent NEB Turbo cells + 2ml of the plasmids.
- The remaining plasmids (8ml) were stored at -20°C.
08.07.2012
Glycerol Stock
| Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name |
|---|---|---|---|---|---|
| | | | | | |
| | | ||||
| | | | | | |
| | | ||||
| | | | | | |
| | |
Gel
We run a gel to control colony PCR results. To do the PCR we use the next primers:
- Forward primer: VF2
- Reverse primer: VR
From left to right:
- Ladder = 5 ml;
- T1 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
- T2 (GFP, BBa_E0040) = 6ml + 1ml of dye = 7ml. Expected size: 720bp
- T3 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
- T4 (pBad, BBa_I13453) = 6ml + 1ml of dye = 7ml. Expected size: 130bp
- Ladder = 5 ml;
Purification (MiniPrep)
| Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name | Concentration, ng/ul |
|---|---|---|---|---|---|---|
| | | | | | | |
| | | | ||||
| | | | | | | |
| | | | ||||
| | | | | | | |
| | | |
07.07.2012
List of parts T1-T6:
| Part | Description | Colony | Name |
|---|---|---|---|
| | | | |
| | | ||
| | | | |
| | | ||
| | | | |
| | |
Start Culture for miniprep and glycerol
- We pick 2 colonies from each plate.
- Add it to 6 ml of LB + Ampicilline (6 tubes: T1, T2, T3, T4, T5, T6).
- Overnight incubation.
Colony PCR of I13453 and E0040
- Gently vortex and briefly centrifuge PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently vortex the samples and spin down.
- When using a thermal cycler that doesn't contain a heated lid, overlay the reaction mixture with 25ul of mineral oil.
- Perform PCR using the recommended thermal cycling conditions outlined below:
| Chemical | Volume |
|---|---|
| | |
| | |
| | |
| | |
| | |
| | |
| Step | Temperature, °C | Time | Number of cycles |
|---|---|---|---|
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
| | | | |
- Comments: LID = 100°C
06.07.2012
Biobrick Retrieval
Biobrick Retrieval Protocol was used. List of retrieval parts:
| Part | Description | Location in a kit | Plasmid Backbone |
|---|---|---|---|
| | | | |
| | | | |
| | | | |
Heat Shock Transformation
- Parts from the list above were transformed using Heat Shock Transformation protocol:
- 20ml of competent NEB Turbo cells + 2ml of the plasmids.
- The remaining plasmids (8ml) were stored at -20°C.
