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Team:BYU Provo/Notebook/CommonProcedures
From 2014.igem.org
(Difference between revisions)
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<h2>TAQ PCR (50 uL Reaction)</h2> | <h2>TAQ PCR (50 uL Reaction)</h2> | ||
| - | <ul><li> | + | <ul><li>35ul ddH20</li> |
| - | <li> | + | <li>10ul 5X REDTAQ buffer (mix well before use!!)</li> |
| - | <li>1. | + | <li>1.0ul 10mM dNTP’s</li> |
| - | <li> | + | <li>1ul each Primer (50 uM stock)</li> |
| - | <li> | + | <li>1ul appropriate diluted template DNA </li> |
| - | <li>Mix well then add 2. | + | <li>Mix well then add 2.5ul REDTAQ polymerase</li></ul> |
<p>Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.</p> | <p>Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.</p> | ||
<ul><li><b>Standard PCR program:</li></b></ul> | <ul><li><b>Standard PCR program:</li></b></ul> | ||
| Line 98: | Line 98: | ||
<h2>Q5 PCR Reaction (50 uL Reaction)</h2> | <h2>Q5 PCR Reaction (50 uL Reaction)</h2> | ||
| - | <ul><li> | + | <ul><li>10uL 5x Q5 Reaction Buffer</li> |
| - | <li> | + | <li>1uL dNTPs</li> |
| - | <li> | + | <li>1uL Forward Primer</li> |
| - | <li> | + | <li>1uL Reverse Primer</li> |
| - | <li> | + | <li>10uL Q5 Enhancer</li> |
| - | <li>23. | + | <li>23.5uL ddH2O</li> |
| - | <li>1- | + | <li>1-2uL Template</li> |
<li>Add ?uL Q5 Polymerase right before you start the PCR reaction.</li></ul> | <li>Add ?uL Q5 Polymerase right before you start the PCR reaction.</li></ul> | ||
<p>Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.</p> | <p>Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.</p> | ||
<h2>Phusion PCR</h2> | <h2>Phusion PCR</h2> | ||
| - | <ul><li> | + | <ul><li>35ul ddH20</li> |
| - | <li> | + | <li>10ul 5X Phusion GC buffer</li> |
| - | <li>1. | + | <li>1.0uL DMSO</li> |
| - | <li>1. | + | <li>1.5ul 10 mM dNTP’s</li> |
| - | <li> | + | <li>1ul each Primer (50 uM stock)</li> |
| - | <li> | + | <li>1ul appropriate diluted template DNA</li> |
| - | <li>0. | + | <li>0.5ul Phusion Polymerase</li></ul> |
<ul><li><b>Standard Phusion PCR program:</li></b></ul> | <ul><li><b>Standard Phusion PCR program:</li></b></ul> | ||
<ol><li>95°C for 2 min</li> | <ol><li>95°C for 2 min</li> | ||
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<h2>Standard 1% Agarose Gel</h2> | <h2>Standard 1% Agarose Gel</h2> | ||
<ul><li>75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt). Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.</li> | <ul><li>75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt). Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.</li> | ||
| - | <li>With gloves on, add 1 drop (~ | + | <li>With gloves on, add 1 drop (~6ul of 1mg/ml) ethidium bromide and swirl to mix.</li> |
<li>Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.</li></ul> | <li>Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.</li></ul> | ||
| + | |||
| + | <h2>Low-melt Agarose Gel</h2> | ||
| + | <ul><li>Mix 0.75g of low-melt agarose with 75mL of TAE. Caution: Low-melt heats more quickly than standard agarose in the microwave.</li> | ||
| + | <li>Add 5ul of ethidium bromide to the agarose solution before casting</li> | ||
| + | <li>Use a large-tooth comb to form wells that will accommodate ~40-50µl of material.</li></ul> | ||
| + | |||
</html> | </html> | ||
Revision as of 01:39, 22 July 2014
BYU 2014 Notebook |
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TAQ PCR (50 uL Reaction)
- 35ul ddH20
- 10ul 5X REDTAQ buffer (mix well before use!!)
- 1.0ul 10mM dNTP’s
- 1ul each Primer (50 uM stock)
- 1ul appropriate diluted template DNA
- Mix well then add 2.5ul REDTAQ polymerase
Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.
- Standard PCR program:
- 95°C for 2 min
- 95°C for 30 sec
- 55°C for 30 sec
- 72°C for 2 min
- Repeat (2-4) 35 times
- 72°C for 5 min
- 4°C for forever
Q5 PCR Reaction (50 uL Reaction)
- 10uL 5x Q5 Reaction Buffer
- 1uL dNTPs
- 1uL Forward Primer
- 1uL Reverse Primer
- 10uL Q5 Enhancer
- 23.5uL ddH2O
- 1-2uL Template
- Add ?uL Q5 Polymerase right before you start the PCR reaction.
Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.
Phusion PCR
- 35ul ddH20
- 10ul 5X Phusion GC buffer
- 1.0uL DMSO
- 1.5ul 10 mM dNTP’s
- 1ul each Primer (50 uM stock)
- 1ul appropriate diluted template DNA
- 0.5ul Phusion Polymerase
- Standard Phusion PCR program:
- 95°C for 2 min
- 95°C for 30 sec
- 55°C for 30 sec
- 72°C for 2 min
- Repeat (2-4) 35 times
- 72°C for 5 min
- 4°C for forever
Standard 1% Agarose Gel
- 75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt). Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.
- With gloves on, add 1 drop (~6ul of 1mg/ml) ethidium bromide and swirl to mix.
- Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.
Low-melt Agarose Gel
- Mix 0.75g of low-melt agarose with 75mL of TAE. Caution: Low-melt heats more quickly than standard agarose in the microwave.
- Add 5ul of ethidium bromide to the agarose solution before casting
- Use a large-tooth comb to form wells that will accommodate ~40-50µl of material.
