Melbourne/Lab BL Notebook/20070916PCR1

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It looks like we have the product ligated into P1010, will need to send off for sequencing.  Not so sure about ligation to RBS

Latest revision as of 15:19, 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

Run on gel

gel1 gel2

gel3

It looks like we have the product ligated into P1010, will need to send off for sequencing. Not so sure about ligation to RBS

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