Griffitts:PCR: Difference between revisions
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<font size="5">PCR with ''Taq'' Polymerase</font> | <font size="5">PCR with ''Taq'' Polymerase</font> | ||
==Procedure== | ==Procedure== | ||
* Prepare your template sample | * Prepare your template sample | ||
** For amplifying ''Sinorhizobium'' sequences, boil a dense suspension of cells in 35 μL [[Griffitts:Common buffers#colony lysis solution (for PCR) (100 mL)|PCR lysis buffer]] for ~2 min and vortex | ** For amplifying ''Sinorhizobium'' sequences, boil a dense suspension of cells in 35 μL [[Griffitts:Common buffers#colony lysis solution (for PCR) (100 mL)|PCR lysis buffer]] for ~2 min and vortex | ||
** For amplifying ''E. coli'', adding cells directly to the reaction is sufficient | ** For amplifying ''E. coli'' with ''Taq'' polymerase (but not ''Pfx50'' polymerase), adding cells directly to the reaction is sufficient | ||
* Thaw the ''Taq'' buffer, dNTPs, and primers | * Thaw the ''Taq'' buffer, dNTPs, and primers | ||
** Keep ''Taq'' polymerase on ice throughout the procedure | ** Keep ''Taq'' polymerase on ice throughout the procedure | ||
Line 15: | Line 13: | ||
** Set the reaction up on ice | ** Set the reaction up on ice | ||
* Select the appropriate cycling program and verify that all of the parameters are correct | * Select the appropriate cycling program and verify that all of the parameters are correct | ||
** Allow 1 minute of extension time per 1 KB DNA being amplified | ** Allow 1 minute of extension time per 1 KB DNA being amplified (15-30 seconds is sufficient for Q5) | ||
* Select "Run program" and select "YES" when asked about heated lid | * Select "Run program" and select "YES" when asked about heated lid | ||
* Wait until block has reached | * Wait until block has reached at least 70°C (this takes a little over 2 min) | ||
* Place PCR tube into the machine | |||
* Lower the lid until it latches and slightly tighten the heated lid onto the tubes | * Lower the lid until it latches and slightly tighten the heated lid onto the tubes | ||
NOTE: When the PCR machine has finished cycling, your samples may be stored at -20°C. You may now proceed to [[Griffitts:Gel_electrophoresis|gel electrophoresis]] and/or [[Griffitts:PCR_clean-up|PCR clean-up]]. | NOTE: When the PCR machine has finished cycling, your samples may be stored at -20°C. You may now proceed to [[Griffitts:Gel_electrophoresis|gel electrophoresis]] and/or [[Griffitts:PCR_clean-up|PCR clean-up]]. | ||
==Reaction recipes== | ==Reaction recipes== | ||
=== | ===20 μL ''Taq'' reactions=== | ||
Use this for diagnostic purposes. | Use this for diagnostic purposes. | ||
{| border="1" cellpadding="5" cellspacing="0" | {| border="1" cellpadding="5" cellspacing="0" | ||
Line 30: | Line 29: | ||
|- | |- | ||
| dH<sub>2</sub>O | | dH<sub>2</sub>O | ||
| | | 14.35 μL | ||
|- | |- | ||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | | [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | ||
| 2. | | 2.0 μL | ||
|- | |- | ||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | | [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | ||
Line 39: | Line 38: | ||
|- | |- | ||
| ''Taq'' polymerase | | ''Taq'' polymerase | ||
| 0. | | 0.15 μL | ||
|- | |- | ||
| forward primer | | forward primer | ||
| 1. | | 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | ||
|- | |- | ||
| reverse primer | | reverse primer | ||
| 1. | | 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | ||
|- | |||
| DNA template | |||
| 1.0 μL | |||
|} | |} | ||
<br> | <br> | ||
===40 μL reactions=== | ===40 μL ''Taq'' reactions=== | ||
This is the standard recipe. Use it in most cases for | This is the standard recipe. Use it in most cases for fragment amplification and sequencing. | ||
{| border="1" cellpadding="5" cellspacing="0" | {| border="1" cellpadding="5" cellspacing="0" | ||
|- | |- | ||
Line 61: | Line 59: | ||
|- | |- | ||
| dH<sub>2</sub>O | | dH<sub>2</sub>O | ||
| | | 28.7 μL | ||
|- | |- | ||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | | [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | ||
| 4 μL | | 4.0 μL | ||
|- | |- | ||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | | [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | ||
| 0 | | 1.0 μL | ||
|- | |- | ||
| ''Taq'' polymerase | | ''Taq'' polymerase | ||
| 0.3 μL | | 0.3 μL | ||
|- | |- | ||
| forward primer | | forward primer | ||
| 2. | | 2.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | ||
|- | |- | ||
| reverse primer | | reverse primer | ||
| 2. | | 2.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | ||
|- | |||
| DNA template | |||
| 2.0 μL | |||
|} | |} | ||
<br> | <br> | ||
Line 92: | Line 89: | ||
|- | |- | ||
| dH<sub>2</sub>O | | dH<sub>2</sub>O | ||
| 27 | | 27 μL | ||
|- | |- | ||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Pfx'' buffer]] | | [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Pfx'' buffer]] | ||
Line 102: | Line 99: | ||
| ''Pfx'' polymerase | | ''Pfx'' polymerase | ||
| 1.0 μL | | 1.0 μL | ||
|- | |||
| forward primer | |||
| 2.4 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| reverse primer | |||
| 2.4 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |- | ||
| DNA template | | DNA template | ||
| 1. | | 2.0 μL | ||
|} | |||
<br> | |||
===20 μL reaction with ''Q5'' polymerase=== | |||
Use this for high-fidelity cloning, especially for products >3kb. | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="120" style="background:#efefef;" | Ingredient | |||
! width="120" style="background:#efefef;" | Per reaction | |||
|- | |||
| dH<sub>2</sub>O | |||
| 12.4 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#5X_Q5_buffer|5X ''Q5'' buffer]] | |||
| 4.0 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | |||
| 0.4 μL | |||
|- | |||
| ''Q5'' polymerase | |||
| 0.2 μL | |||
|- | |- | ||
| forward primer | | forward primer | ||
| | | 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | ||
|- | |- | ||
| reverse primer | | reverse primer | ||
| | | 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | ||
|- | |||
| DNA template | |||
| 1.0 μL | |||
|} | |} | ||
<br> | <br> | ||
===40 μL reaction with ''Q5'' polymerase=== | |||
Use this for high-fidelity cloning, especially for products >3kb. | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="120" style="background:#efefef;" | Ingredient | |||
! width="120" style="background:#efefef;" | Per reaction | |||
|- | |||
| dH<sub>2</sub>O | |||
| 24.8 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#5X_Q5_buffer|5X ''Q5'' buffer]] | |||
| 8.0 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | |||
| 0.8 μL | |||
|- | |||
| ''Q5'' polymerase | |||
| 0.4 μL | |||
|- | |||
| forward primer | |||
| 2.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| reverse primer | |||
| 2.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| DNA template | |||
| 2.0 μL | |||
|} | |||
<br> | <br> | ||
Line 119: | Line 175: | ||
|- | |- | ||
! width="200" style="background:#efefef;" | Ingredient | ! width="200" style="background:#efefef;" | Ingredient | ||
! width="150" style="background:#efefef;" | | ! width="150" style="background:#efefef;" | 20 μL reaction | ||
with ''Taq'' polymerase | with ''Taq'' polymerase | ||
! width="150" style="background:#efefef;" | 40 μL reaction | ! width="150" style="background:#efefef;" | 40 μL reaction | ||
Line 125: | Line 181: | ||
! width="150" style="background:#efefef;" | 40 μL reaction | ! width="150" style="background:#efefef;" | 40 μL reaction | ||
with ''Pfx'' polymerase | with ''Pfx'' polymerase | ||
! width="150" style="background:#efefef;" | 40 μL reaction | |||
with ''Q5'' polymerase | |||
|- | |- | ||
| dH<sub>2</sub>O | | dH<sub>2</sub>O | ||
| | | 16.15 μL | ||
| | | 32.3 μL | ||
| | | 31.3 μL | ||
| 24.8 μL | |||
|- | |- | ||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | | [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | ||
| 2. | | 2.0 μL | ||
| 4.0 μL | | 4.0 μL | ||
| 4.0 μL | | 4.0 μL | ||
| 8.0 μL | |||
|- | |- | ||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | | [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | ||
| 0.5 μL | | 0.5 μL | ||
| 1.0 μL | |||
| 1.2 μL | |||
| 0.8 μL | | 0.8 μL | ||
|- | |- | ||
| polymerase | | polymerase | ||
| 0. | | 0.15 μL | ||
| 0.3 μL | | 0.3 μL | ||
| 1.0 μL | | 1.0 μL | ||
| 0.4 μL | |||
|- | |- | ||
| forward primer | | forward primer | ||
| 0. | | 0.1 μL ''undiluted'' | ||
| 0. | | 0.2 μL ''undiluted'' | ||
| 0. | | 0.25 μL ''undiluted'' | ||
| 0.2 μL ''undiluted'' | |||
|- | |- | ||
| reverse primer | | reverse primer | ||
| 0. | | 0.1 μL ''undiluted'' | ||
| 0. | | 0.2 μL ''undiluted'' | ||
| 0. | | 0.25 μL ''undiluted'' | ||
| 0.2 μL ''undiluted'' | |||
|- | |- | ||
| '''Total volume (without | | '''Total volume (without template)''' | ||
| ''' | | '''19 μL''' | ||
| ''' | | '''38 μL''' | ||
| '''38 | | '''38 μL''' | ||
| '''38 μL''' | |||
|} | |} | ||
<br> | <br> |
Latest revision as of 16:02, 30 September 2013
PCR with Taq Polymerase
Procedure
- Prepare your template sample
- For amplifying Sinorhizobium sequences, boil a dense suspension of cells in 35 μL PCR lysis buffer for ~2 min and vortex
- For amplifying E. coli with Taq polymerase (but not Pfx50 polymerase), adding cells directly to the reaction is sufficient
- Thaw the Taq buffer, dNTPs, and primers
- Keep Taq polymerase on ice throughout the procedure
- Combine components according to one of the recipes below
- Set the reaction up on ice
- Select the appropriate cycling program and verify that all of the parameters are correct
- Allow 1 minute of extension time per 1 KB DNA being amplified (15-30 seconds is sufficient for Q5)
- Select "Run program" and select "YES" when asked about heated lid
- Wait until block has reached at least 70°C (this takes a little over 2 min)
- Place PCR tube into the machine
- Lower the lid until it latches and slightly tighten the heated lid onto the tubes
NOTE: When the PCR machine has finished cycling, your samples may be stored at -20°C. You may now proceed to gel electrophoresis and/or PCR clean-up.
Reaction recipes
20 μL Taq reactions
Use this for diagnostic purposes.
Ingredient | Per reaction |
---|---|
dH2O | 14.35 μL |
10X Taq buffer | 2.0 μL |
dNTPs | 0.5 μL |
Taq polymerase | 0.15 μL |
forward primer | 1.0 μL diluted 1:10 |
reverse primer | 1.0 μL diluted 1:10 |
DNA template | 1.0 μL |
40 μL Taq reactions
This is the standard recipe. Use it in most cases for fragment amplification and sequencing.
Ingredient | Per reaction |
---|---|
dH2O | 28.7 μL |
10X Taq buffer | 4.0 μL |
dNTPs | 1.0 μL |
Taq polymerase | 0.3 μL |
forward primer | 2.0 μL diluted 1:10 |
reverse primer | 2.0 μL diluted 1:10 |
DNA template | 2.0 μL |
40 μL reaction with Pfx polymerase
Use this for high-fidelity cloning.
Ingredient | Per reaction |
---|---|
dH2O | 27 μL |
10X Pfx buffer | 4.0 μL |
dNTPs | 1.2 μL |
Pfx polymerase | 1.0 μL |
forward primer | 2.4 μL diluted 1:10 |
reverse primer | 2.4 μL diluted 1:10 |
DNA template | 2.0 μL |
20 μL reaction with Q5 polymerase
Use this for high-fidelity cloning, especially for products >3kb.
Ingredient | Per reaction |
---|---|
dH2O | 12.4 μL |
5X Q5 buffer | 4.0 μL |
dNTPs | 0.4 μL |
Q5 polymerase | 0.2 μL |
forward primer | 1.0 μL diluted 1:10 |
reverse primer | 1.0 μL diluted 1:10 |
DNA template | 1.0 μL |
40 μL reaction with Q5 polymerase
Use this for high-fidelity cloning, especially for products >3kb.
Ingredient | Per reaction |
---|---|
dH2O | 24.8 μL |
5X Q5 buffer | 8.0 μL |
dNTPs | 0.8 μL |
Q5 polymerase | 0.4 μL |
forward primer | 2.0 μL diluted 1:10 |
reverse primer | 2.0 μL diluted 1:10 |
DNA template | 2.0 μL |
Master Mix Calculations
Ingredient | 20 μL reaction
with Taq polymerase |
40 μL reaction
with Taq polymerase |
40 μL reaction
with Pfx polymerase |
40 μL reaction
with Q5 polymerase |
---|---|---|---|---|
dH2O | 16.15 μL | 32.3 μL | 31.3 μL | 24.8 μL |
10X Taq buffer | 2.0 μL | 4.0 μL | 4.0 μL | 8.0 μL |
dNTPs | 0.5 μL | 1.0 μL | 1.2 μL | 0.8 μL |
polymerase | 0.15 μL | 0.3 μL | 1.0 μL | 0.4 μL |
forward primer | 0.1 μL undiluted | 0.2 μL undiluted | 0.25 μL undiluted | 0.2 μL undiluted |
reverse primer | 0.1 μL undiluted | 0.2 μL undiluted | 0.25 μL undiluted | 0.2 μL undiluted |
Total volume (without template) | 19 μL | 38 μL | 38 μL | 38 μL |
Primer Dilution
- 27 μL dH2O
- 3 μL primer
Repeat for both primers