Melbourne/Lab BL Notebook/PCR1

From 2007.igem.org

(Difference between revisions)
Line 6: Line 6:
! PCR mix
! PCR mix
! PCR program
! PCR program
 +
! [[Melbourne/Lab_BL_Notebook/PrimersI|Primer]] II
|-
|-
|5ul 10x buffer\\
|5ul 10x buffer\\
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2.5ul MgSO4 (Supplied in PCR kit)
2.5ul MgSO4 (Supplied in PCR kit)
-
1.5ul Primer I (10uM)
+
1.5ul Primer [[Melbourne/Lab_BL_Notebook/PrimersI#Oligo_15|BL_FP1_s]] (10uM stock)
-
1.5ul Primer II (10uM)
+
1.5ul Primer II (10uM stock)
1ul Template ([[Melbourne/pJS010|pJS010]])
1ul Template ([[Melbourne/pJS010|pJS010]])
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4°C forever
4°C forever
 +
|-
 +
|Reaction A => Primer BL_Con1_as
 +
 +
Reaction B => Primer BL_Con2_as
 +
 +
Reaction C => Primer BL_Con3_as
 +
 +
Reaction D => Primer BL_Con4_as
 +
 +
Reaction E => Primer BL_Con5_as
 +
 +
Reaction F => Primer BL_Con6_as
 +
 +
Reaction G => Primer BL_Con7_as
|-
|-
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|}
|}
 +
=Protocol for PCR reactions 1-7=
 +
For amplification of the kinase domain of ComP
 +
 +
{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 +
|-
 +
! PCR mix
 +
! PCR program
|-
|-
-
====PCR mix====                                         
 
|5ul 10x buffer\\
|5ul 10x buffer\\
-
5ul Enhancer
 
0.6ul dNTPs (25mM stock)
0.6ul dNTPs (25mM stock)
Line 50: Line 70:
2.5ul MgSO4 (Supplied in PCR kit)
2.5ul MgSO4 (Supplied in PCR kit)
-
1.5ul Primer I (10uM)
+
1.5ul Primer I (10uM stock)
-
1.5ul Primer II (10uM)
+
1.5ul Primer VR (10uM stock)
1ul Template ([[Melbourne/pJS010|pJS010]])
1ul Template ([[Melbourne/pJS010|pJS010]])
Line 59: Line 79:
32.5ul ddH<sub>2</sub>O
32.5ul ddH<sub>2</sub>O
 +
|94°C -  5'
-
---
+
94°C  - 30"
-
50ul Total
+
59°C  - 30"
-
|
+
 
 +
68°C  - 35" (goto step 2 x30)
 +
 
 +
68°C  - 1.5'
 +
 
 +
4°C forever
|-
|-
 +
 +
| 50ul Total
 +
|}

Revision as of 15:10, 10 October 2007

Protocol for PCR reactions A~G

For amplifying the photoreceptor and transmembrane domains of NpSopII-NpHtrII.

PCR mix PCR program Primer II
5ul 10x buffer\\

5ul Enhancer

0.6ul dNTPs (25mM stock)

2.5ul MgSO4 (Supplied in PCR kit)

1.5ul Primer BL_FP1_s (10uM stock)

1.5ul Primer II (10uM stock)

1ul Template (pJS010)

0.4ul Pfx Platinum (Invitrogen)

32.5ul ddH2O

94°C - 5'

94°C - 30"

59°C - 30"

68°C - 35" (goto step 2 x30)

68°C - 1.5'

4°C forever

Reaction A => Primer BL_Con1_as

Reaction B => Primer BL_Con2_as

Reaction C => Primer BL_Con3_as

Reaction D => Primer BL_Con4_as

Reaction E => Primer BL_Con5_as

Reaction F => Primer BL_Con6_as

Reaction G => Primer BL_Con7_as

50ul Total

Protocol for PCR reactions 1-7

For amplification of the kinase domain of ComP

PCR mix PCR program
5ul 10x buffer\\


0.6ul dNTPs (25mM stock)

2.5ul MgSO4 (Supplied in PCR kit)

1.5ul Primer I (10uM stock)

1.5ul Primer VR (10uM stock)

1ul Template (pJS010)

0.4ul Pfx Platinum (Invitrogen)

32.5ul ddH2O

94°C - 5'

94°C - 30"

59°C - 30"

68°C - 35" (goto step 2 x30)

68°C - 1.5'

4°C forever

50ul Total
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