Griffitts:PCR: Difference between revisions
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</div> | </div> | ||
<font size="5">PCR with Taq Polymerase</font> | <font size="5">PCR with ''Taq'' Polymerase</font> | ||
==Procedure== | ==Procedure== | ||
# Thaw buffer, | * 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 ''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 [[Griffitts:TaqPCR#Reaction recipes|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 [[Griffitts:Gel_electrophoresis|gel electrophoresis]] and/or [[Griffitts:PCR_clean-up|PCR clean-up]]. | |||
==Reaction recipes== | |||
===20 μL ''Taq'' reactions=== | |||
Use this for diagnostic purposes. | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="120" style="background:#efefef;" | Ingredient | |||
! width="120" style="background:#efefef;" | Per reaction | |||
|- | |||
| dH<sub>2</sub>O | |||
| 14.35 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | |||
| 2.0 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | |||
| 0.5 μL | |||
|- | |||
| ''Taq'' polymerase | |||
| 0.15 μL | |||
|- | |||
| forward primer | |||
| 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| reverse primer | |||
| 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| DNA template | |||
| 1.0 μL | |||
|} | |||
<br> | |||
===40 μL ''Taq'' reactions=== | |||
This is the standard recipe. Use it in most cases for fragment amplification and sequencing. | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="120" style="background:#efefef;" | Ingredient | |||
! width="120" style="background:#efefef;" | Per reaction | |||
|- | |||
| dH<sub>2</sub>O | |||
| 28.7 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]] | |||
| 4.0 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | |||
| 1.0 μL | |||
|- | |||
| ''Taq'' polymerase | |||
| 0.3 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> | |||
===40 μL reaction with ''Pfx'' polymerase=== | |||
Use this for high-fidelity cloning. | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="120" style="background:#efefef;" | Ingredient | |||
! width="120" style="background:#efefef;" | Per reaction | |||
|- | |||
| dH<sub>2</sub>O | |||
| 27 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Pfx'' buffer]] | |||
| 4.0 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]] | |||
| 1.2 μL | |||
|- | |||
| ''Pfx'' polymerase | |||
| 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 | |||
| 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 | |||
| 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| reverse primer | |||
| 1.0 μL [[Griffitts:TaqPCR#Primer Dilution|diluted 1:10]] | |||
|- | |||
| DNA template | |||
| 1.0 μL | |||
|} | |||
<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> | |||
== | ===Master Mix Calculations=== | ||
''' | {| border="1" cellpadding="5" cellspacing="0" | ||
|- | |||
! width="200" style="background:#efefef;" | Ingredient | |||
! width="150" style="background:#efefef;" | 20 μL reaction | |||
0. | with ''Taq'' polymerase | ||
! width="150" style="background:#efefef;" | 40 μL reaction | |||
with ''Taq'' polymerase | |||
! width="150" style="background:#efefef;" | 40 μL reaction | |||
''' | with ''Pfx'' polymerase | ||
! width="150" style="background:#efefef;" | 40 μL reaction | |||
with ''Q5'' polymerase | |||
|- | |||
| 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]] | |||
| 2.0 μL | |||
| 4.0 μL | |||
| 4.0 μL | |||
| 8.0 μL | |||
|- | |||
| [[Griffitss:Stock_solutions#10_mM_DNTPs|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''' | |||
|} | |||
<br> | |||
<br> | |||
== | ==Primer Dilution== | ||
* | * 27 μL dH<sub>2</sub>O | ||
* | * 3 μL primer | ||
Repeat for both primers |
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