Team:METU Turkey/Results Discussion/Transformation

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Revision as of 21:01, 27 October 2010

MAIN STEPS/TIME TABLE
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- Fresh o/n bacteria
- LB Agar plate preparation[30 min]
- Transformation [2.5-3.0 h]
- Incubation of plates [14-17 h]

MATERIALS
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Heat Block
Incubator
p10, p100 and p1000

SOC medium
Ice
Ice box
Bent
desultory
Parafilm
Spreader
LB Agar plates with antibiotic
Vector DNA
Competent cells

CHECK-LIST PROCEDURE
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- Check availability of LB Agar
- Check availability of SOC medium and preheat
- Check presence of sterile dH2O
- Check incubator with shaker and heat block to be worked properly

- Mix (spin) 1 uL vector DNA and 50 uL competent cells.(1-10 ng vector)
- Incubate in ice bath for 30 min.
- Heat shock for 20 sec. in 42C at heatblock.(not exceed 30 sec)
- Ice bath for 30 sec.
- Add 600ul of SOC medium on bacteria culture.
- Incubate in 200 rpm shaker for 1h
- Inoculate into plates

SOLUTIONS
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Preparation of LB
- 4 gr LB Broth
- 2.7 gr Agar
- 200 mL dH2O
- Autoclave the bottle.
- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic).
- Pour the plates .
- Keep the plates +4 C

Preparation of Commercial LB Agar (LB agar-Xgal- IPTG-Amp/Fermentas)
- Mix 200 ml dH2O + 1 paket LB agar
- Heat in Microwave 360 watt; 6-8 min
- Pour to plate

Preparation of SOC medium (1L)
- 900 mL dH2O
- 20g Bacto tryptone extract
- 5g Bacto yeast extract
- 2 mL of 5M NaCl
- 2.5 mL of 1M KCl
- 10 mL of 1M MgCl2
- 10 mL of 1M MgSO
- For maximum effectiveness, the SOC medium should be adjusted to a pH of 7.0
- Autoclave the solution
- Add 20 mL of 1M glucose (autoclaved solution)
- Adjust to 1L with dH2O

NOTES
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- Time intervals of heat shock and ice bath should be strictly obeyed.
- For the competent cells and vector DNA, inoculation to media must be performed to either 1.5 mL or 2.0 mL eppendorfs.
- Always bacteria should be added onto the vector solution.
- Media should be added into the bacteria solution(not bacteria on media).
- Incubation at heat shock must not exceed 30 seconds.
- Preheating of plates to 37 C before inoculation is recommended(20 min)
- Competent cells should be thawed in ice
- All eppendorfs should be cold before addition of vector and bacteria.
- Shaking of eppendorfs in the incubator must be horizontal (not vertical)

TROUBLESHOOTING
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- If transformation is not completed, firstly control the competent cells whether they are working or not. If competent cells are working, control whether the plasmid concentration is enough or not (10 ng plasmid is enough).

Low transformation efficiency or low colony number
- Ensure that excess mineral oil is not transferred into the transformation reaction when pipetting the enzyme-treated DNA. Using the smallest pipet tips available, insert the pipet tip completely below the mineral layer overlay and clear the pipet tip while submerged beneath the mineral oil overlay before collecting the sample.
- Ensure that sufficient mutant DNA is synthesized in the reaction. Increase the amount of the enzym-treated DNA used in the transformation reaction to 4 uL.
- Visualize the DNA template on a gel to verify the quantity and quality. Nicked or linearized plasmid DNA will not generate complete circular product. Verify that the template DNA is at least 80% supercoiled.
- It is not uncommon to observe low numbers of colonies, especially when generating large mutations. Most of the colonies that do appear, however, will contain mutagenized plasmid.

Low mutagenesis efficiency or low colony number with the control reaction
- Ensure that supercompetent cells are stored at the bottom of a –80 C freezer immediately upon arrival (see also Transformation Guidelines).
- Verify that the agar plates were prepared correctly.
- For best visualization of the blue (beta-gal+) phenotype, the control plates must be incubated for at least 16 h at 37 C.

Transformation efficiency is too low (evaluated in control transformation with supercoiled DNA)
- Old bacterial cultures were used to prepare competent cells.
- Seed overnight culture from a freshly streaked bacterial culture plate. Refresh bacterial strains weekly. For seeding of overnight E.coli DH5αlpha culture, use only <24 hours fresh culture plates.

Low number or no transformants
- Volume of ligation reaction mixture too large.
- Do not use more than 5 uL of ligation reaction mixture per 50 uL of competent cells.

DNA amount is too high
- Do not use more than 100 ng of plasmid DNA for transformation of 50 uL of competent cells.

Inefficient ligation
- Simple sticky end ligation reactions should yield 50-200 colonies. If the efficiency of competent cells was acceptable, but transformation of ligation mixture yielded no or only few transformants, repeat cloning experiment.
- Use high quality DNA, enzymes and follow the recommended ligation protocol.
- Background colonies without plasmid.
- Insufficient amount of antibiotic in agar medium.
- Use recommended amount of appropriate antibiotic in LB agar plates. Antibiotics are heat-sensitive therefore allow the LB medium to cool to 55 C before addition of the antibiotic to it.

Satellite colonies
- Some fast growing strains (e.g. C600) lead to formation of smaller satellite colonies around transformants after
>16 hours of incubation. Use shorter incubation times and do not involve such small colonies into clone analysis.