Difference between revisions of "Team:Linkoping Sweden/Experiments"
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| − | + | Transformation - Double Heat Shock | |
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| − | + | Method | |
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| − | <ul> | + | <ul> |
| − | <li> | + | <li>Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.</li> |
| − | <li>Pipette | + | <li>Label 1.5 ml tubes with part name or well location.</li> |
| − | <li> | + | <li>Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.</li> |
| − | <li>Pick | + | <li>Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.</li> |
| − | + | <li>Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.</li> | |
| + | <li>Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.</li> | ||
| + | <li>Close 1.5 ml tubes, incubate on ice for 3 h: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.</li> | ||
| + | <li>Heat shock tubes at 42°C for 45 sec: Timing is critical.</li> | ||
| + | Incubate on ice for 2 min: Return transformation tubes to ice bucket.</li> | ||
| + | <li>Heat shock tubes again at 42°C for 45 sec: Timing is critical.</li> | ||
| + | <li>Pipette 250 µL LB media to each transformation</li> | ||
| + | <li>Incubate at 37°C for 1 hours, shaking at 170 rpm</li> | ||
| + | <li>Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.</li> | ||
| + | <li>Spin down cells at 6800 g for 3 mins and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.</li> | ||
| + | <li>Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.</li> | ||
| + | <li>Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.</li> | ||
| + | <li>Count colonies for control transformation: Count colonies on the 100 μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.</li> | ||
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Revision as of 13:29, 15 September 2018
Experiment protocols
DNA Kit Plate Instructions
To use the DNA in the Distribution Kit, follow these instructions: Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µL
- With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
- Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
- Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
- Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours. Use the resulting culture to miniprep the DNA AND make your own glycerol stock.
Transformation - Double Heat Shock
Method
- Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
- Label 1.5 ml tubes with part name or well location.
- Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
- Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
- Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
- Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
- Close 1.5 ml tubes, incubate on ice for 3 h: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
- Heat shock tubes at 42°C for 45 sec: Timing is critical. Incubate on ice for 2 min: Return transformation tubes to ice bucket.
- Heat shock tubes again at 42°C for 45 sec: Timing is critical.
- Pipette 250 µL LB media to each transformation
- Incubate at 37°C for 1 hours, shaking at 170 rpm
- Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
- Spin down cells at 6800 g for 3 mins and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
- Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
- Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
- Count colonies for control transformation: Count colonies on the 100 μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.