Melbourne/Lab BL Notebook/20070916PCR1

From 2007.igem.org

(Difference between revisions)
Line 10: Line 10:
|5ul 10x buffer\\
|5ul 10x buffer\\
-
5ul Enhancer
+
5ul 10x Enhancer
0.6ul dNTPs (25mM stock)
0.6ul dNTPs (25mM stock)
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1ul Template ([[Melbourne/pJS010|pJS010]])
1ul Template ([[Melbourne/pJS010|pJS010]])
-
0.4ul Pfx Platinum (Invitrogen)
+
0.4ul Pfx  
32.5ul ddH<sub>2</sub>O
32.5ul ddH<sub>2</sub>O
Line 93: Line 93:
4°C forever
4°C forever
-
|Reaction A => Primer BL_Con1_s
+
|Reaction 1 => Primer BL_Con1_s
-
Reaction B => Primer BL_Con2_s
+
Reaction 2 => Primer BL_Con2_s
-
Reaction C => Primer BL_Con3_s
+
Reaction 3 => Primer BL_Con3_s
-
Reaction D => Primer BL_Con4_s
+
Reaction 4 => Primer BL_Con4_s
-
Reaction E => Primer BL_Con5_s
+
Reaction 5 => Primer BL_Con5_s
-
Reaction F => Primer BL_Con6_s
+
Reaction 6 => Primer BL_Con6_s
-
Reaction G => Primer BL_Con7_s
+
Reaction 7 => Primer BL_Con7_s
|-
|-
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==Gel Purification of PCR products A~G and 1~7==
==Gel Purification of PCR products A~G and 1~7==
-
PCR product of the expected size was excised from .8% agarose gel and gel purified using the Invitrogen gel purification kit. Protocol as detailed in the kit.
+
PCR product of the expected size was excised from .8% agarose gel and gel purified using the Invitrogen gel purification kit. Protocol as detailed in the kit.  
 +
 
 +
=Second Round PCR=
 +
For stitching products A~G to 1~7. Gel purified PCR products from the above reaction were used.
 +
 
 +
{| 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
 +
! <Reaction, TemplateI, TemplateII>
 +
|-
 +
|5ul 10x buffer\\
 +
 
 +
2.5ul 10x Enhancer
 +
 
 +
0.6ul dNTPs (25mM stock)
 +
 
 +
2.5ul MgSO4 (Supplied in PCR kit)
 +
 
 +
1.5ul Primer BL_FP1_s (10uM stock)
 +
 
 +
1.5ul Primer VR (10uM stock)
 +
 
 +
5ul Template I {A~G}
 +
 
 +
5ul Template II {1-7}
 +
 
 +
0.4ul Pfx
 +
 
 +
27ul ddH<sub>2</sub>O
 +
|94°C -  5'
 +
 
 +
94°C  - 1'
 +
 
 +
50°C  - 1'
 +
 
 +
68°C  - 3' (goto step 2 x30)
 +
 
 +
68°C  - 10'
 +
 
 +
4°C forever
 +
 
 +
|<Reaction A1, A, 1>
 +
 
 +
<Reaction B2, B, 2>
 +
 
 +
<Reaction C3, C, 3>
 +
 
 +
<Reaction D4, D, 4>
 +
 
 +
<Reaction E5, E, 5>
 +
 
 +
<Reaction F6, F, 6>
 +
 
 +
<Reaction G7, G, 7>
 +
|-
 +
 
 +
| 50ul Total
 +
|}

Revision as of 15:29, 10 October 2007

Contents

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 10x 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 (Some non-specific bands)

Reaction B => Primer BL_Con2_as

Reaction C => Primer BL_Con3_as (Some non-specific bands)

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

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 1 => Primer BL_Con1_s

Reaction 2 => Primer BL_Con2_s

Reaction 3 => Primer BL_Con3_s

Reaction 4 => Primer BL_Con4_s

Reaction 5 => Primer BL_Con5_s

Reaction 6 => Primer BL_Con6_s

Reaction 7 => Primer BL_Con7_s

50ul Total

Gel Purification of PCR products A~G and 1~7

PCR product of the expected size was excised from .8% agarose gel and gel purified using the Invitrogen gel purification kit. Protocol as detailed in the kit.

Second Round PCR

For stitching products A~G to 1~7. Gel purified PCR products from the above reaction were used.

PCR mix PCR program <Reaction, TemplateI, TemplateII>
5ul 10x buffer\\

2.5ul 10x Enhancer

0.6ul dNTPs (25mM stock)

2.5ul MgSO4 (Supplied in PCR kit)

1.5ul Primer BL_FP1_s (10uM stock)

1.5ul Primer VR (10uM stock)

5ul Template I {A~G}

5ul Template II {1-7}

0.4ul Pfx

27ul ddH2O

94°C - 5'

94°C - 1'

50°C - 1'

68°C - 3' (goto step 2 x30)

68°C - 10'

4°C forever

<Reaction A1, A, 1>

<Reaction B2, B, 2>

<Reaction C3, C, 3>

<Reaction D4, D, 4>

<Reaction E5, E, 5>

<Reaction F6, F, 6>

<Reaction G7, G, 7>

50ul Total
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