Team:Imperial College/PCR

From 2008.igem.org

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(New page: {{Imperial/StartPage2}} __NOTOC__ ==PCR== This protocol is desgined for use with the stratagene ''PfuUltra'' II Fusion DNA polymerase and is based in part on the ''PfuUltra'' II Fusion D...)
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{{Imperial/StartPage2}}
{{Imperial/StartPage2}}
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__NOTOC__
 
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==PCR==
 
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{{Imperial/Box1|PCR|
This protocol is desgined for use with the stratagene ''PfuUltra'' II Fusion DNA polymerase and is based in part on the ''PfuUltra'' II Fusion DNA polymerase usage manual. [http://www.stratagene.com/manuals/600670.pdf ''PfuUltra'' II Fusion Manual]
This protocol is desgined for use with the stratagene ''PfuUltra'' II Fusion DNA polymerase and is based in part on the ''PfuUltra'' II Fusion DNA polymerase usage manual. [http://www.stratagene.com/manuals/600670.pdf ''PfuUltra'' II Fusion Manual]
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======Aim======
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To produce clones of two genes from ''B.subtilis'' that are too big to have synthesised by GeneArt; for use as an integration site and gene knockout (''epsE'') or for their original purpose as a transcriptional regulator (''xylR'').
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===Aim===
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To produce clones of sequences from vectors; for use as integration sites (''amyE''), antibiotic resistance (Spectinomycin - ''aad9'') and as a transcriptional repressor (''lacI'').
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To produce clones of two genes from ''B.subtilis'' that are too big to have synthesised by GeneArt; for use as an integration site and gene knockout (EpsE) or for their original purpose as a transcriptional regulator (XylR).
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To produce clones of sequences from vectors; for use as integration sites (AmyE), antibiotic resistance (Spectinomycin) and as a transcriptional repressor (LacI).
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A modified protocol for using Taq polymerase can be used if less fidelity is required
A modified protocol for using Taq polymerase can be used if less fidelity is required
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======Equipment======
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===Equipment===
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Heated lid PCR machine
Heated lid PCR machine
Thin walled PCR tube
Thin walled PCR tube
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======Reagents======
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*Distilled H<sub>2</sub>O: 18.75μL
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*10Χ ''PfuUltra™''II reaction buffer: 2.5μL
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*dNTP mix (10mM): 0.5μL
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*''B.subtilis'' genomic DNA (100ng/μL): 1μL
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*Forward Primer (100ng/μL): 1μL
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*Reverse Primer (100ng/μL): 1μL
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*''PfuUltra®'' II fusion HS DNA polymerase: 0.25μL
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===Reagents===
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''Total Reaction Volume: 25μL''
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{| border="1" style="text-align:center"
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|-
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! Reagent !! Volume
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|-
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|| Distilled H<sub>2</sub>O || 18.75μL
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|-
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|| 10Χ ''PfuUltra™''II reaction buffer || 2.5μL
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|-
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|| dNTP mix (10mM) || 0.5μL
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|-
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|| ''B.subtilis'' genomic DNA (100ng/μL) || 1μL
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|-
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|| Forward Primer (100ng/μL) || 1μL
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|-
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|| Reverse Primer (100ng/μL) || 1μL
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|-
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|| ''PfuUltra®'' II fusion HS DNA polymerase || 0.25μL
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|-
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|| Total Reaction Volume || 25μL
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|-
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|}
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Note: Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 100ng of DNA should be added and the volume of H<sub>2</sub>O to be added should be adjusted to maintain a reaction volume of 25μL.
Note: Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 100ng of DNA should be added and the volume of H<sub>2</sub>O to be added should be adjusted to maintain a reaction volume of 25μL.
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The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence.
The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence.
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======Protocol======
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===Protocol===
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Add all the reagents in order (down the list) sequentially to the PCR tube, mxing after each addition.
Add all the reagents in order (down the list) sequentially to the PCR tube, mxing after each addition.
Place the PCR tubes into the PCR machine and set the programme to the following set-up:
Place the PCR tubes into the PCR machine and set the programme to the following set-up:
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{|
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#Initial Denaturation: 30 seconds at 95°C (longer for genomic DNA)
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|-
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#10 Cycles of: 30 second denaturation at 95°C
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| Initial Denaturation: || 30 seconds at 95°C (longer for genomic DNA)
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#30 second annealing time at Primer T<sub>m</sub> - ~3°C (complementary section of primer T<sub>m</sub>)
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#15 seconds (+ 15 second for each additional kb) extending time at 68C (30 seconds for genomic templates)
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| 10 Cycles of: || 30 second denaturation at 95°C
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#20 - 30 Cycles of: 30 second denaturation at 95°C
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|-
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#30 second annealing time at Primer T<sub>m</sub> - ~3°C (total primer T<sub>m</sub>)
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| || 30 second annealing time at Primer T<sub>m</sub> - ~3°C (complementary section of primer T<sub>m</sub>)
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#15 seconds (+ 15 second for each additional kb)extending time at 68°C (30 seconds for genomic templates)
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|-
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#Final Extension: 5 minutes at 68°C
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| || 15 seconds (+ 15 second for each additional kb) extending time at 68C (30 seconds for genomic templates)
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|-
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| 20 - 30 Cycles of: || 30 second denaturation at 95°C
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|-
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| || 30 second annealing time at Primer T<sub>m</sub> - ~3°C (total primer T<sub>m</sub>)
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|-
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| || 15 seconds (+ 15 second for each additional kb)extending time at 68°C (30 seconds for genomic templates)
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|Final Extension: || 5 minutes at 68°C
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The resulting solution can then be purified using a PCR purification column, by gel electrophoresis followed by spin purification or can simply be ligated ready for use.
The resulting solution can then be purified using a PCR purification column, by gel electrophoresis followed by spin purification or can simply be ligated ready for use.
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|}}
{{Imperial/EndPage|Protocols|}}
{{Imperial/EndPage|Protocols|}}

Revision as of 12:06, 28 October 2008


PCR

This protocol is desgined for use with the stratagene PfuUltra II Fusion DNA polymerase and is based in part on the PfuUltra II Fusion DNA polymerase usage manual. [http://www.stratagene.com/manuals/600670.pdf PfuUltra II Fusion Manual]

Aim

To produce clones of two genes from B.subtilis that are too big to have synthesised by GeneArt; for use as an integration site and gene knockout (epsE) or for their original purpose as a transcriptional regulator (xylR).

To produce clones of sequences from vectors; for use as integration sites (amyE), antibiotic resistance (Spectinomycin - aad9) and as a transcriptional repressor (lacI).

A modified protocol for using Taq polymerase can be used if less fidelity is required

Equipment

Heated lid PCR machine

Thin walled PCR tube

Reagents
  • Distilled H2O: 18.75μL
  • 10Χ PfuUltra™II reaction buffer: 2.5μL
  • dNTP mix (10mM): 0.5μL
  • B.subtilis genomic DNA (100ng/μL): 1μL
  • Forward Primer (100ng/μL): 1μL
  • Reverse Primer (100ng/μL): 1μL
  • PfuUltra® II fusion HS DNA polymerase: 0.25μL

Total Reaction Volume: 25μL

Note: Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 100ng of DNA should be added and the volume of H2O to be added should be adjusted to maintain a reaction volume of 25μL.

If a vector is used as the template, 5ng of plasmid DNA should be used instead and the volume of H2O to be added should be adjusted accordingly.

If PCR is proving difficult, particularly for denaturation, DMSO can be added to a final concentration of 10% to increase efficiency

The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence.

Protocol

Add all the reagents in order (down the list) sequentially to the PCR tube, mxing after each addition.

Place the PCR tubes into the PCR machine and set the programme to the following set-up:

  1. Initial Denaturation: 30 seconds at 95°C (longer for genomic DNA)
  2. 10 Cycles of: 30 second denaturation at 95°C
  3. 30 second annealing time at Primer Tm - ~3°C (complementary section of primer Tm)
  4. 15 seconds (+ 15 second for each additional kb) extending time at 68C (30 seconds for genomic templates)
  5. 20 - 30 Cycles of: 30 second denaturation at 95°C
  6. 30 second annealing time at Primer Tm - ~3°C (total primer Tm)
  7. 15 seconds (+ 15 second for each additional kb)extending time at 68°C (30 seconds for genomic templates)
  8. Final Extension: 5 minutes at 68°C

The resulting solution can then be purified using a PCR purification column, by gel electrophoresis followed by spin purification or can simply be ligated ready for use.



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