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Team:DTU-Denmark/Methods/PCR-mastermix
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(→1x recipe for mastermix to a 50uL PCR reaction) |
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===1x recipe for mastermix to a 50uL PCR reaction=== | ===1x recipe for mastermix to a 50uL PCR reaction=== | ||
*'''10uL HF buffer''' | *'''10uL HF buffer''' | ||
| - | **HF buffer should be 5x | + | **HF buffer should be 5x or adjust volume so final conc. of HF is 1x. |
*'''1uL dNTP's''' | *'''1uL dNTP's''' | ||
**This can vary depending on conc. the final conc. should be 0.2mM. | **This can vary depending on conc. the final conc. should be 0.2mM. | ||
| - | *3uL of forward primer | + | *'''3uL of forward primer''' |
| - | *3uL fo reverse primer | + | *'''3uL fo reverse primer''' |
**The primer volume can differ quite a lot depending on which conc. you have them in. The final conc. of the each primer should be in the range of 1uM to 0.1uM but this can also be hard to generalize. | **The primer volume can differ quite a lot depending on which conc. you have them in. The final conc. of the each primer should be in the range of 1uM to 0.1uM but this can also be hard to generalize. | ||
| - | *0.5uL of polymerase | + | *'''0.5uL of polymerase''' |
**This again depend on the unit per microliter conc. of the polymerase. Use approx. 2.5U per reaction. | **This again depend on the unit per microliter conc. of the polymerase. Use approx. 2.5U per reaction. | ||
| - | *1uL template | + | *'''1uL template''' |
**The conc. of template can vary and this can also be one of the variables to turn on if the PCR goes wrong. | **The conc. of template can vary and this can also be one of the variables to turn on if the PCR goes wrong. | ||
| - | *31.5uL MQ-water | + | *'''31.5uL MQ-water''' |
**If using more or less of the previous reactant then just adjust the MQ amount so that the final volume will be 50uL | **If using more or less of the previous reactant then just adjust the MQ amount so that the final volume will be 50uL | ||
Revision as of 08:20, 27 June 2013
Mastermix for PCR
A master mix lessens the work when making PCR-reactions by mixing all the common ingredients in one tube and then distributing it to the different samples. It saves time, pipette tips and minimize the pipetting errors.
1x recipe for mastermix to a 50uL PCR reaction
- 10uL HF buffer
- HF buffer should be 5x or adjust volume so final conc. of HF is 1x.
- 1uL dNTP's
- This can vary depending on conc. the final conc. should be 0.2mM.
- 3uL of forward primer
- 3uL fo reverse primer
- The primer volume can differ quite a lot depending on which conc. you have them in. The final conc. of the each primer should be in the range of 1uM to 0.1uM but this can also be hard to generalize.
- 0.5uL of polymerase
- This again depend on the unit per microliter conc. of the polymerase. Use approx. 2.5U per reaction.
- 1uL template
- The conc. of template can vary and this can also be one of the variables to turn on if the PCR goes wrong.
- 31.5uL MQ-water
- If using more or less of the previous reactant then just adjust the MQ amount so that the final volume will be 50uL
