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Team:DTU Denmark/protocols
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Revision as of 13:39, 21 October 2009
Digestion with restriction enzymes. 5
Miniprep for plasmid purification. 7
DNA purification from enzymatic reaction. 9
Transformation of chemically competent E. coli 10
High Efficiency Yeast Transformation. 11
Gel electrophoreses
Agarose
1X TAE buffer
MiliQ water
Ethidium Bromide 0.625 mg/ml (Amresco)
Mini Sub Cell GT (BioRad)
Hyperladder I (Bioline) (Appendix 6.1.1)
5x DNA Loading Buffer, Blue (Bioline)
Preparation of 1% agarose gel For a final volume of 500 ml, 5 g of agarose was dissolved into 500 ml of TAE buffer and mixed with a magnetic stirrer. The agarose was melted in microwave for 5 min. The agarose is completely melted when the solution is totally clear (no veil).
Preparation of DNA Loading buffer was added to DNA. When mixing small amounts this can be done on a piece of parafilm.
Agarose gel electrophoresis Capacity of small wells app.: ~ 15 μl, big wells: ~ 50μl.. Standard 75 mV 400 mA 1 hour / 45 min. Standard amount of hyperladder loaded: 5 μl (4 μl hyperladder 1 μl loading buffer).
Imaging the gel Gel Doc 2000 (Bio Rad). Quantity One 4.0.2 (Bio Rad). The exposure varies.
HyperladderTM I
HyperladderTM I was used throughout the study as ladder for all gels
PCR amplification
The following recipe was used for amplifying genes used for cloning with the Phusion-polymerase. If more than one PCR-reaction was made, a mastermix containing the polymerase, dNTP, PCR-buffer and water could be made giving more precise concentrations in the mix.
It is critical that the DNA Polymerase is the last component added to the PCR mixture, since the enzyme exhibits 3´→5´ exonuclease activity that can degrade primers in the absence of dNTPs.
When running PCR reactions with Taq the PCR buffer is 10x so only 5μl needs to be added
Phusion Polymerase has the highest fidelity of any commercial thermo stable polymerase (50X greater than Taq). This is the reason that this enzyme was used for all cloning procedures described in this report. Taq is mostly used for colony PCR where the purpose is to detect the presence of a certain sequence and not to use the PCR product for further cloning.
|
T [ºC] |
Time |
|
|
|
98 |
30 |
sec |
|
|
98 |
30 |
sec |
Repeat 30 times |
|
57* |
30 |
sec | |
|
72 |
15-60** |
sec | |
|
72 |
8 |
min |
|
|
12 |
Hold |
|
|
Table 2: Standard PCR program with Phusion polymerase.
* for primers > 20nt, anneal for 10 – 30 seconds at a Tm +3°C of the lower Tm primer. for primers ≤ 20nt, use an annealing temperature equal to the Tm of the lower Tm primer for calculating of melting temperature see Primer design. **15 sec per kb for low complexity DNA (e.g. plasmid, lambda or BAC DNA). 30 sec per kb for high complexity genomic DNA
|
T [ºC] |
Time |
|
|
|
94 |
2 |
min |
|
|
94 |
30 |
sec |
Repeat 30 times |
|
56* |
30 |
sec | |
|
72 |
4** |
min | |
|
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