From 2007.igem.org

< Melbourne | Lab BL Notebook(Difference between revisions)

Latest revision as of 15:19, 11 October 2007

1 Protocol for PCR reactions A~G
2 Protocol for PCR reactions 1-7
- 2.1 Gel Purification of PCR products A~G and 1~7
3 Second Round PCR
- 3.1 Gel purification of second round PCR product
4 Digestion/Ligation of Second Round PCR product
- 4.1 Ligation of constructs into Death/RBS plasmid
- 4.2 Confirmation Digest

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

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

@@ Line 176: / Line 176: @@
 All the reactions had large smears, but we cut out a band around 2.3kb (expected size) and gel purified as above.
-=Digestion of Second Round PCR product=
+=Digestion/Ligation of Second Round PCR product=
 Per PCR reaction:
+{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+|-
+! Per 2° PCR {A1~G7}
+|-
+|6ul purified DNA {A1~G7}
-ul purified DNA
+ul 10x NEB Buffer 2
+ul 10x BSA
+ul XbaI
+ul PstI
+ul H<sub>2</sub>O
+|-
+| 20ul Total
+|} Incubated at 37°C for 45', then at RT for 6hrs. Heat inactivate at 80°C for 10'.
+Also...
+{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+|-
+! BBa_J61035 (S/P)
+|-
+|5ul [http://partsregistry.org/Part:BBa_J61035 BBa_J61035]
+ul 10x NEB Buffer 2
+ul 10x BSA
+ul SpeI
+ul PstI
+ul H<sub>2</sub>O
+|-
+| 20ul Total
+|}
+And...
+{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+|-
+! BBa_P1010 (X/P)
+|-
+|5ul [http://partsregistry.org/Part:BBa_P1010 BBa_P1010](AmpR)
 ul 10x NEB Buffer 2
@@ Line 189: / Line 236: @@
 ul PstI
-ul H<sub>2<\sub>O
+ul H<sub>2</sub>O
+|-
+| 20ul Total
+|}
+The latter two reactions were gel purified and the following ligations were set up:
+====Ligation of constructs into Death/RBS plasmid====
+{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+|-
+! Per 2° PCR {A1~G7}
+|-
+|5ul X/P digested DNA {A1~G7}
+ul X/P digested BBa_P1010
+ul 2x Quick ligase buffer
+ul T4 Ligase
+|-
+| 20ul Total
+|}
+{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+|-
+! Per 2° PCR {A1~G7}
+|-
+|5ul X/P digested DNA {A1~G7}
+ul S/P digested BBa_J61035
+ul 2x Quick ligase buffer
+ul T4 Ligase
+|-
+| 20ul Total
+|}
+Incubated at RT for 2hrs then [[Melbourne/Transformation_Protocol|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====
+{| border="2" cellpadding="4" cellspacing="0" style="margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+|-
+! Per miniprepped sample
+|-
+|5ul miniprepped sample
+ul 10x NEB Buffer 2
+ul 10x BSA
+.17ul XbaI
+.17ul PstI
+.5ul H<sub>2</sub>O
+|-
+| 20ul Total
+|} Incubate at 37°C  for 2 1hr
+Run on gel
+[[Image:Melbourne 20070920 bl1a.jpg|400px|Thumb|gel1]]
+[[Image:Melbourne_Igem20070920_gel2.jpg|400px|Thumb|gel2]]
+[[Image:Melbourne20070920_bl3a.jpg |400px|Thumb|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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Melbourne/Lab BL Notebook/20070916PCR1

From 2007.igem.org

Latest revision as of 15:19, 11 October 2007

Contents

Protocol for PCR reactions A~G

Protocol for PCR reactions 1-7

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

Second Round PCR

Gel purification of second round PCR product

Digestion/Ligation of Second Round PCR product

Ligation of constructs into Death/RBS plasmid

Confirmation Digest