Team:DTU-Denmark/PCR protocol

From 2011.igem.org

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(Created page with "== PCR protocol == PCR reaction components: a. Enzyme b. Forward primer c. Reverse primer d. dNTPs e. template DNA f. buffer g. MilliQ or distilled water Amounts per one react...")
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Amounts per one reaction (100μl)
Amounts per one reaction (100μl)
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Taq+ pFU (μl) Phusion (μl)
+
{| class="wikitable"
-
enzyme 0,5 0,5
+
|-
-
Forward primer 2,5 5
+
! Reagent
-
Reverse primer 2,5 5
+
! TAQ + PFU [μl]
-
dNTP 4 4
+
! Phusion [μl]
-
Template 1 1
+
|-
-
Buffer 10 20
+
| Enzyme
-
water 79,5 64,5
+
| 0.5
 +
| 0.5
 +
|-
 +
| Forward primer
 +
| 2.5
 +
| 5
 +
|-
 +
| Reverse primer
 +
| 2.5
 +
| 5
 +
|-
 +
| dNTP
 +
| 4
 +
| 4
 +
|-
 +
| Template
 +
| 1
 +
| 1
 +
|-
 +
| Buffer
 +
| 10
 +
| 20
 +
|-
 +
| Water
 +
| 79.5
 +
| 64.5
 +
|}
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Master mix
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== Master mix ==
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a. Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes.
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<ol style="list-style-type:lower-alpha">
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b. Master mix should contain dNTPs, buffer and water.
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  <li> Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes.
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Remember to put in following order:
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  <li> Master mix should contain what all reactions has in common
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1. Master mix
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  <li> Enzyme should be added last
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2. Primers
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</ol>
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3. Template DNA
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Remember to add reagents in the following order
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4. Enzyme
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# Master mix
 +
# (Primers)
 +
# (Template DNA)
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Remember primers should be diluted 1:10.
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Remember primers should be diluted 1:10 from stock.
Phusion is used if we want to have better proof-reading.  
Phusion is used if we want to have better proof-reading.  
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In order to estimate size and amount of DNa fragments look below:
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In order to estimate size and amount of DNA fragments look below:
   
   
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Estimating the size of sample:
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== Estimating the amount of DNA in the sample: ==
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We may look at the band intensities and from proportion and amount of DNA given in the picture below calculate the concentration of our DNA sample.??
+
By comparing the band intensities of the ladder you can estimate the concentration of DNA in your sample.

Revision as of 15:02, 18 September 2011

PCR protocol

PCR reaction components: a. Enzyme b. Forward primer c. Reverse primer d. dNTPs e. template DNA f. buffer g. MilliQ or distilled water

Amounts per one reaction (100μl)

Reagent TAQ + PFU [μl] Phusion [μl]
Enzyme 0.5 0.5
Forward primer 2.5 5
Reverse primer 2.5 5
dNTP 4 4
Template 1 1
Buffer 10 20
Water 79.5 64.5

Master mix

  1. Prepare master mix for the amount of your PCR reactions +3 more. Always prepare more, because of the errors of the pipettes.
  2. Master mix should contain what all reactions has in common
  3. Enzyme should be added last

Remember to add reagents in the following order

  1. Master mix
  2. (Primers)
  3. (Template DNA)

Remember primers should be diluted 1:10 from stock. Phusion is used if we want to have better proof-reading. In order to estimate size and amount of DNA fragments look below:

Estimating the amount of DNA in the sample:

By comparing the band intensities of the ladder you can estimate the concentration of DNA in your sample.

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