Melbourne/Lab BL Notebook/20070916PCR1

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Revision as of 15:17, 11 October 2007

Contents

Protocol for PCR reactions A~G

For amplifying the photoreceptor and transmembrane domains of NpSopII-NpHtrII. All -ve controls were clean.

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

Gel purification of second round PCR product

All the reactions had large smears, but we cut out a band around 2.3kb (expected size) and gel purified as above.

Digestion/Ligation of Second Round PCR product

Per PCR reaction:

Per 2° PCR {A1~G7}
6ul purified DNA {A1~G7}

2ul 10x NEB Buffer 2

2ul 10x BSA

1ul XbaI

1ul PstI

8ul H2O

20ul Total
Incubated at 37°C for 45', then at RT for 6hrs. Heat inactivate at 80°C for 10'.

Also...

BBa_J61035 (S/P)
5ul [http://partsregistry.org/Part:BBa_J61035 BBa_J61035]

2ul 10x NEB Buffer 2

2ul 10x BSA

1ul SpeI

1ul PstI

9ul H2O

20ul Total

And...

BBa_P1010 (X/P)
5ul [http://partsregistry.org/Part:BBa_P1010 BBa_P1010](AmpR)

2ul 10x NEB Buffer 2

2ul 10x BSA

1ul XbaI

1ul PstI

9ul H2O

20ul Total

The latter two reactions were gel purified and the following ligations were set up:

Ligation of constructs into Death/RBS plasmid

Per 2° PCR {A1~G7}
5ul X/P digested DNA {A1~G7}

4ul X/P digested BBa_P1010

10ul 2x Quick ligase buffer

1ul T4 Ligase

20ul Total
Per 2° PCR {A1~G7}
5ul X/P digested DNA {A1~G7}

4ul S/P digested BBa_J61035

10ul 2x Quick ligase buffer

1ul T4 Ligase

20ul Total

Incubated at RT for 2hrs then transformed into competent DH5-alpha. Also included some 5minute ligation incubation controls => 5 minute ligations are significantly (~5x) more efficient than 2 hours.

Pick 2 colonies per death plasmid ligation, and 4 colonies per RBS ligation and culture in LB+Amp for 12hrs.

Miniprep (nuclease free water elution)

Confirmation Digest

Per miniprepped sample
5ul miniprepped sample

1ul 10x NEB Buffer 2

1ul 10x BSA

.17ul XbaI

.17ul PstI

2.5ul H2O

20ul Total
Incubate at 37°C for 2 1hr

gel1 gel2 gel3

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