IGEM:Paris Bettencourt 2012/Notebook/RE group/Lab notes: Difference between revisions
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==23.07.2012== | ==23.07.2012== | ||
=== PCR of K175027 and K175041 === | === PCR of K175027 and K175041 === | ||
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We made a PCR using 1uL of DNA from these 2 tubes. The protocol was exactly the same then the one described just below. | We made a PCR using 1uL of DNA from these 2 tubes. The protocol was exactly the same then the one described just below. | ||
===Gel of miniPrep digestion I13453 and R0011=== | ===Gel of miniPrep digestion I13453 and R0011=== | ||
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===Gel of PCR BBa_I13453 (pBad) and BBa_R0011 (pLac)=== | ===Gel of PCR BBa_I13453 (pBad) and BBa_R0011 (pLac)=== | ||
[[Image:IGEM_Paris_RG_PCR_gel_23_07_2012.gif|thumb|300px|right|gel_23_07_2012.gif ]] | |||
=== PCR of miniprep BBa_I13453 (pBad) and BBa_R0011 (pLac) === | === PCR of miniprep BBa_I13453 (pBad) and BBa_R0011 (pLac) === |
Revision as of 12:08, 23 July 2012
23.07.2012
PCR of K175027 and K175041
We received these parts from anotehr igem team
K175027: it is the restriction site for IsceI. Size: 30bp K175041: pLacI controlled IsceI homing endonuclease generator (IsceI ha an LVA degradation tag). Expected size: 1114bp
We made a PCR using 1uL of DNA from these 2 tubes. The protocol was exactly the same then the one described just below.
Gel of miniPrep digestion I13453 and R0011
Digestion of miniPrep I13453 and R0011
Gel of PCR BBa_I13453 (pBad) and BBa_R0011 (pLac)
PCR of miniprep BBa_I13453 (pBad) and BBa_R0011 (pLac)
Since all our gels for these parts were inconsistant, put the sequencing was consistent, we decided to make yet anotehr gel, hence the PCR.
Biobrick name | Taken from tube | Name of PCR tube | Size of fragment |
---|---|---|---|
BBa_I13453 (pBAD) | pRG 003 | 1 | |
BBa_I13453 (pBAD) | pRG 004 | 2 | |
BBa_R0011 (pLac) | pRG 005 | 3 | |
BBa_R0011 (pLac) | pRG 006 | 4 |
- Gently mix PCR Master Mix (2X) after thawing.
- Place a thin-walled PCR tube on ice and add the following components for each 50ul reaction:
- Gently mix the samples.
- Perform PCR using the recommended thermal cycling conditions outlined below:
Chemical | Volume |
---|---|
Step | Temperature, °C | Time | Number of cycles |
---|---|---|---|
- Comments: LID = 100°C
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 BBa_K098991, BBa_K093012 and BBa_B0032
- 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;
13.07.2012
Purification (MiniPrep)
Part | Description | Plasmid Backbone | Size, bp | Colony | Stock name | Concentration, ng/ul |
---|---|---|---|---|---|---|
Glycerol stock
- We make two tubes per liquid culture tube. One that will be stored at -20°C and another one that will be stored at -80°C
- In each tube we added:
- 750uL of solution from the culture tube
- 250 uL of glycerol 60%
Biobrick name | Colony | Tube name |
---|---|---|
BBa_E0240 | 1 | pRG007 |
BBa_E0240 | 2 | pRG008 |
BBa_K117004 | 1 | pRG009 |
BBa_K117004 | 2 | pRG010 |
BBa_I746902 | 1 | pRG011 |
BBa_I746902 | 2 | pRG012 |
BBa_J004450 | 1 | pRG013 |
BBa_J004450 | 2 | pRG014 |
BBa_I13540 | 1 | pRG015 |
BBa_I13540 | 2 | pRG016 |
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.
- 6 mL of LB + Ampicilline (for E0240, K117004, I746902, I13540, I13540, Control+ R0011)
- 6 mL of LB + Kan (for J04450)
- Incubate over night
Gel
We made a 1% agarose gel
- Mix 50mL of BET (0.5%) with 0.5g of agarose
- Put in the microwave 1.30min and then an additional 20s (till the solution is transparent)
- Wait a little bit till it cools down (no more steam)
- Pull in a big plate (to make a big size gel)
- Wait 30min
- For each PCR tube: Mix 1uL of die with 5uL of solution from the PCR tube
- Put this mix in gel wells
- Wait for migration (approx 30min). Voltage: 100V
- Put the gel in an ethidium bromide bath for 10min
- Rince in H2O destain for 5min
- Take picture
From left to right, we've put the following parts in well:
- Ladder (1kb)
- E0240 colony 1 (pcr tube 1)
- E02410 colony 2 (pcr tube 2)
- Ladder 1kB
- K117004 colony 1 (pcr tube 3)
- K117004 colony 2 (pcr tube 4)
- I746902 colony 1 (pcr tube 5)
- I746902 colony 2 (pcr tube 6)
- J04450 colony 1 (pcr tube 7)
- J04450 colony 2 (pcr tube 8)
- I13540 colony 1 (pcr tube 9)
- I13540 colony 2 (pcr tube 10)
- Control+ R0011 colony 1 (pcr tube 11)
- Control+ R0011 colony 2 (pcr tube 12)
- K117004 colony 1 (pcr tube 3)
- Ladder 1kB
Gel Analysis: We do not see any parts. Something probably went wrong with the PCR. To check that our parts are well expressed, and are the correct parts we will tcheck it in another way: digestion of our minipreped plasmids and then gel (see 14.07.2012)
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
Transformation results
Successful transformation:
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 BBa_I13453 and BBa_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.