Melbourne/Lab BL Notebook/PCR1

From 2007.igem.org

< Melbourne | Lab BL Notebook(Difference between revisions)
 
(4 intermediate revisions not shown)
Line 25: Line 25:
32.5ul ddH<sub>2</sub>O
32.5ul ddH<sub>2</sub>O
 +
|94°C -  5'
|94°C -  5'
94°C  - 30"
94°C  - 30"
-
59°C  - 30"
+
59°C  - 35"
-
68°C  - 35" (goto step 2 x30)
+
68°C  - 1.5' (goto step 2 x30)
-
68°C  - 1.5'
+
68°C  -10'
4°C forever
4°C forever
Line 53: Line 54:
| 50ul Total
| 50ul Total
-
}
+
|}
=Protocol for PCR reactions 1-7=
=Protocol for PCR reactions 1-7=
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! PCR mix
! PCR mix
! PCR program
! PCR program
 +
! [[Melbourne/Lab_BL_Notebook/PrimersI|PrimerI]]
|-
|-
|5ul 10x buffer\\
|5ul 10x buffer\\
Line 83: Line 85:
94°C  - 30"
94°C  - 30"
-
59°C  - 30"
+
59°C  - 35"
-
68°C  - 35" (goto step 2 x30)
+
68°C  - 1.5' (goto step 2 x30)
-
68°C  - 1.5'
+
68°C  - 10'
4°C forever
4°C forever
 +
 +
|Reaction A => Primer BL_Con1_s
 +
 +
Reaction B => Primer BL_Con2_s
 +
 +
Reaction C => Primer BL_Con3_s
 +
 +
Reaction D => Primer BL_Con4_s
 +
 +
Reaction E => Primer BL_Con5_s
 +
 +
Reaction F => Primer BL_Con6_s
 +
 +
Reaction G => Primer BL_Con7_s
|-
|-
| 50ul Total
| 50ul Total
|}
|}

Latest revision as of 15:15, 10 October 2007

Protocol for PCR reactions A~G

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

PCR mix PCR program PrimerII
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 - 35"

68°C - 1.5' (goto step 2 x30)

68°C -10'

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 PrimerI
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 - 35"

68°C - 1.5' (goto step 2 x30)

68°C - 10'

4°C forever

Reaction A => Primer BL_Con1_s

Reaction B => Primer BL_Con2_s

Reaction C => Primer BL_Con3_s

Reaction D => Primer BL_Con4_s

Reaction E => Primer BL_Con5_s

Reaction F => Primer BL_Con6_s

Reaction G => Primer BL_Con7_s

50ul Total
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