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/Protocols
From 2009.igem.org
Contents |
Cloning
Our cloning cycle consists of two days: a light day (not too much to do) and a heavy day (lots to do).
- #Colony selecting and checking (light day)
- Plasmid extraction and digestion (first part of heavy day).
- Gel extraction/Purification, ligation, and transformation (second part of heavy day).
Colony selecting and checking
- Preparation
- Circle the selected n colonies on the plate.
- Prepare a checking plate (divide the plate into n sections)
- Prepare enough 4ml LB in 10ml tubes (depending on how many cultures you want).
- Prepare at least n+2 PCR tubes (2=positive+negative)
- Action
- For each colony: pick the selected colony off (using a tip or toothpick) then:
- Dip it in the PCR solution.
- Dip it in the liquid (optional).
- Streak it on the checking plate.
- End of action
- Put the checking plate and the liquid cultures into the incubator for 12-16hrs.
- Put the PCR tubes in the PCR machine and run.
- When the PCR is done, run it on gel electrophoresis then
- Mark the colonies (on the checking plate) which are correct and incorrect.
- For each colony: pick the selected colony off (using a tip or toothpick) then:
Plasmid extraction and digestion
- For plasmid extraction, we use Viogen's miniprep kit. This usually takes around an hour.
- For digestion using restriction enzymes, we follow the protocol provided (we use different brands). Then we put it at 37C for various amounts of time ranging from 1/2hr to a few hours, the average being around 2hrs.
Gel extraction/purification, ligation, and transformation
- Depending on the digestion, we either perform gel extraction or just directly purify it.
- For that, we use [Someone's] Gel extraction and PCR purification kits respectively.
- Ligation is performed following the provided by the manufacturer (again, we use different brands). Usually we leave it in the cupboard (about 22C-25C) for about 17mins before doing transformation. Depending on the difficulty of the ligation, we also ligate overnight at 4C.
- We transform using [someone's] 5 minute transformation protocol, then incubated at 37C for 12-16hrs. Depending on the origins of the plasmid we're transforming, we use different amounts of competent cells. Amounts:
- 25uL: is usually sufficient.
- 16-20uL: For those that grow a lot of colonies.
- 33uL: For transforming straight from the biobrick plates.
- 50uL: For transforming biobricks that are difficult to transform.
"Magic" Colony PCR
Magic PCR protocol:
| 50ul (x1) | x10 | |
|---|---|---|
| template | 1 | |
| VR+VF2 | 2 | 20 |
| dNTP | 2 | 20 |
| 10x buffer | 5 | 50 |
| taq | 0.2 | 2 |
| ddH20 | 39.8 | 398 |
| total | 50 | 490 |
Ideal for 19 or 23 PCR tubes.
- For 19 tubes -> take 24.5ul/tube (24.5ul wasted on tips)
- For 23 tubes -> take 20ul/tube (30ul wasted on tips)
23 tubes (21 colonies + positive and negative control) is ideal since it fits snugly on a 25-well gel, and also one plate can be divided into 24.
Reporting Assay
Day -2:
- Plates streaked to produce single colonies.
- Plates grown overnight (12-16hrs) at 37C.
Day -1:
- For each part to test, three 3ml cultures of LB and ampicillin (10mM) were inoculated with single colonies from the freshly streaked plates (three biological replicates) in a 10ml tube.
- Cultures were grown overnight (12-16hrs) at 37C shaking at 180-200rpm.
Day 0:
- The cultures were diluted to 0.0325 OD600 into 3-5ml (usually 3ml) and grown for 1hr at 37C shaking at 180-200rpm (keep the shaking speed consistant throughout.)
- For every 1hr for 5 or 6 hours, do:
- Take out the liquid cultures
- For all liquid cultures for, do:
- Extract 250ml into a cuvette that is able to measure the OD600 with only 250ml.
- After measuring the OD600, extract 200 and put it into a well on a black 96-well plate.
- Put the liquid cultures back into the incubator and let the continue growing (note the time taken from taking out the liquid culture to putting it back. This time should be kept as consistant as possible).
- Measure the Fluorescence.
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