Exeter/11 July 2013

From 2013.igem.org

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(Digestions)
(NanoDrop and Digestions)
 
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=Morning=
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{{:Team:Exeter/Template/Header}}
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<div class="container">
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    <div class="row">
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        <div class="span" style="text-align:justify">
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Today we will be trying to digest and ligate some of our BioBricks for the first time!
Today we will be trying to digest and ligate some of our BioBricks for the first time!
-
'''MiniPrep'''
+
=== MiniPrep ===
We extracted the plasmids for...
We extracted the plasmids for...
-
- BBa_K608002, a promoter and RBS
+
* [http://parts.igem.org/Part:BBa_K608002 BBa_K608002], a promoter and RBS
-
- BBa_K592005, our FixJ protein intermediate
+
-
- BBa_K592004, our blue light sensor
+
-
- BBa_B0015, a terminator
+
-
The normal MiniPrep protocol was followed, however the BBa_K608002 and BBa_B0015 Bricks only had 2/3rds as much genetic material collected as the other Bricks. (Plasmids collected from 2 Eppendorfs, as opposed to the usual 3)
+
* [http://parts.igem.org/Part:BBa_K592005 BBa_K592005], our FixJ protein intermediate
-
=NanoDrop and Digestions=
+
* [http://parts.igem.org/Part:BBa_K592012 BBa_K592004], our blue light sensor
 +
 
 +
* [http://parts.igem.org/Part:BBa_K592012 BBa_B0015], a terminator
 +
 
 +
The normal MiniPrep protocol was followed, however the BBa_K608002 and BBa_B0015 Bricks only had 2/3 as much genetic material collected as the other Bricks. (Plasmids collected from 2 Eppendorfs, as opposed to the usual 3)
 +
 
 +
==NanoDrop and Digestions==
We are combining...
We are combining...
Line 20: Line 27:
- a promoter and RBS to the blue light sensor (BBa_K608002 + BBa_K592004)
- a promoter and RBS to the blue light sensor (BBa_K608002 + BBa_K592004)
-
- a promoter and RBS to the FixJ protein coding region (BBa_K608002 + BBa_K592004)
+
- a promoter and RBS to the FixJ protein coding region (BBa_K608002 + BBa_K592005)
- the magenta pigment to a terminator (BBa_K592012 + BBa_B0015)
- the magenta pigment to a terminator (BBa_K592012 + BBa_B0015)
Line 32: Line 39:
For our promoter and RBS Brick (BBa_K608002) and terminator Brick (BBa_B0015) we have two replicates.
For our promoter and RBS Brick (BBa_K608002) and terminator Brick (BBa_B0015) we have two replicates.
-
- Promoter and RBS #1 - 29.1ng/ul
+
- Promoter and RBS #1 - 29.1 ng/&micro;l
-
- Promoter and RBS #2 - 25.2ng/ul
+
- Promoter and RBS #2 - 25.2ng/&micro;l
-
- Terminator #1 - 27.6ng/ul
+
- Terminator #1 - 27.6ng/&micro;l
-
- Terminator #2 - 19.2ng/ul
+
- Terminator #2 - 19.2ng/&micro;l
-
- Blue light sensor - 38.3ng/ul
+
- Blue light sensor - 38.3ng/&micro;l
-
- FixJ intermediate - 58.1ng/ul
+
- FixJ intermediate - 58.1ng/&micro;l
-
- Yellow pigment - 45.7ng/ul
+
- Yellow pigment - 45.7ng/&micro;l
-
- Magenta pigment - 72.0ng/ul
+
- Magenta pigment - 72.0ng/&micro;l
-
- RFP control - 43.4ng/ul
+
- RFP control - 43.4ng/&micro;l
-
'''To get a concentration of 25ng/ul for the restriction digest, we will use...'''
+
'''To get a concentration of 25ng/&micro;l for the restriction digest, we will use...'''
-
- Promoter and RBS #1 - 17.18ul DNA and 25.82ul distilled water
+
- Promoter and RBS #1 - 17.18 &micro;l DNA and 25.82 &micro;l distilled water
-
- Promoter and RBS #2 - 19.84ul DNA and 23.16ul distilled water
+
- Promoter and RBS #2 - 19.84 &micro;l DNA and 23.16 &micro;l distilled water
-
- Terminator #1 - 18.12ul DNA and 24.88ul distilled water
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- Terminator #1 - 18.12 &micro;l DNA and 24.88 &micro;l distilled water
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- Terminator #2 - 26.04ul DNA and 16.96ul distilled water
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- Terminator #2 - 26.04 &micro;l DNA and 16.96 &micro;l distilled water
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- Blue light sensor - 13.05ul DNA and 24.95ul distilled water
+
- Blue light sensor - 13.05 &micro;l DNA and 24.95 &micro;l distilled water
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- FixJ intermediate - 8.61ul DNA and 34.39ul distilled water
+
- FixJ intermediate - 8.61 &micro;l DNA and 34.39 &micro;l distilled water
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- Yellow pigment - 10.94ul DNA and 32.06ul distilled water
+
- Yellow pigment - 10.94 &micro;l DNA and 32.06&micro;l distilled water
-
- Magenta pigment - 6.94ul DNA and 36.06ul distilled water
+
- Magenta pigment - 6.94 &micro;l DNA and 36.06 &micro;l distilled water
-
- RFP control - 11.52ul DNA and 31.48ul distilled water
+
- RFP control - 11.52 &micro;l DNA and 31.48 &micro;l distilled water
-
The buffer we are using contains BSA already, so the change from the iGEM Digestion protocol is that 0.5ul BSA has been replaced with 0.5ul distilled water, which is included in the values above.
+
The buffer we are using contains BSA already, so the change from the iGEM Digestion protocol is that 0.5 &micro;l BSA has been replaced with 0.5 &micro;l distilled water, which is included in the values above.
'''Restriction digest'''
'''Restriction digest'''
Line 82: Line 89:
-Labelled PCR tubes with Part A, Part B, pSB1K3 and RFP control (Parts A and B described above)
-Labelled PCR tubes with Part A, Part B, pSB1K3 and RFP control (Parts A and B described above)
-
- Add correct amounts of distilled water and DNA (see "To get a concentration of 25ng/ul for the restriction digest, we will use...")
+
- Add correct amounts of distilled water and DNA (see "To get a concentration of 25 ng/&micro;l for the restriction digest, we will use...")
-
- Add 5ul of 10X FastDigest buffer (which contains BSA) to each tube.
+
- Add 5 &micro;l of 10X FastDigest buffer (which contains BSA) to each tube.
-
- To the Part A tubes, add 1ul EcoRI and 1ul SpeI.
+
- To the Part A tubes, add 1 &micro;l <i>EcoRI</i> and 1 &micro;l <i>SpeI</i>.
-
- To the Part B tubes, add 1ul XbaI and 1ul PstI.
+
- To the Part B tubes, add 1 &micro;l <i>XbaI</i> and 1 &micro;l <i>PstI</i>.
-
- To the pSB1K3 tube, add 1ul EcoRI and 1ul PstI.
+
- To the pSB1K3 tube, add 1 &micro;l <i>EcoRI</i> and 1 &micro;l <i>PstI</i>.
-
- To the RFP control, add 1ul EcoRI and 1ul PstI.
+
- To the RFP control, add 1 &micro;l <i>EcoRI</i> and 1 &micro;l <i>PstI</i>.
- Mix all by pipetting up and down gently. DO NOT VORTEX. Flick the tubes to collect the liquid at the bottom.
- Mix all by pipetting up and down gently. DO NOT VORTEX. Flick the tubes to collect the liquid at the bottom.
-
- Incubate in a thermocycler - 37oC for 30 minutes, then 80oC for 20 minutes. As a precaution, the final temperature was set at 4oC, incase we didn't get to the thermocycler immediately when the cycle finished.
+
- Incubate in a thermocycler - 37 &deg;C for 30 minutes, then 80 &deg;C for 20 minutes. As a precaution, the final temperature was set at a 4 &deg;C infinite hold, incase we didn't get to the thermocycler immediately when the cycle finished.
 +
 
 +
==Ligation==
 +
 
 +
We need...
 +
 
 +
- PCR tubes
 +
 
 +
- distilled water
 +
 
 +
- T4 DNA ligase reaction buffer
 +
 
 +
- T4 DNA ligase (MUST be kept in the freezer until last minute)
 +
 
 +
- pSB1K3 from digest
 +
 
 +
- Part A from digest
 +
 
 +
- Part B from digest
 +
 
 +
- RFP control from digest
 +
 
 +
Method:
 +
 
 +
- Label a PCR tube as "New Part" (New Part 1 is promoter and RBS + blue light sensor, New Part 2 is promoter and RBS + FixJ intermediate, New Part 3 is magenta pigment + terminator, New Part 4 is yellow pigment + terminator)
 +
 
 +
- To these tubes, add 2 &micro;l pSB1K3, 3.3 &micro;l Part A, 3.9 &micro;l Part B, 1.0 &micro;l T4 DNA ligase reaction buffer and 0.5 &micro;l T4 DNA ligase.
 +
 
 +
- Mix gently by pipetting up and down. DO NOT VORTEX.
 +
 
 +
- Also label a PCR tubes as ligation control. This will contain 2ul from the RFP control digest, 6.5 &micro;l purite water, 1 &micro;l T4 DNA ligase reaction buffer and 0.5 &micro;l T4 DNA ligase. Mixed gently by pipetting up and down.
 +
 
 +
- Collect liquid at bottom of all PCR tubes by flicking.
 +
 
 +
- Incubate in the thermocycler for 30 minutes at 16 &deg;C then 20 minutes at 80 &deg;C. As a precaution, the final temperature was set at a 4 &deg;C infinite hold, incase we didn't get to the thermocycler immediately when the cycle finished.
 +
 
 +
Our products from ligation were stored overnight at -20 &deg;C to be worked with tomorrow.
-
=Ligation=
+
Take me back to the [https://2013.igem.org/Team:Exeter/Notebook notebook].
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=Ligate=
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  </div>
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* K592010 and B0015
+
</div>
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* K592012 and B0015
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{{:Team:Exeter/Template/Footer}}
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* K592003 and Q04510
+
-
* K592005 and B0015
+
-
* K592002 and B0015
+
-
* K592001 and B0015
+
-
* K592004 and B0015
+

Latest revision as of 20:00, 2 October 2013

Exeter iGEM 2013 · Paint by Coli


Today we will be trying to digest and ligate some of our BioBricks for the first time!

MiniPrep

We extracted the plasmids for...

  • [http://parts.igem.org/Part:BBa_K608002 BBa_K608002], a promoter and RBS
  • [http://parts.igem.org/Part:BBa_K592005 BBa_K592005], our FixJ protein intermediate
  • [http://parts.igem.org/Part:BBa_K592012 BBa_K592004], our blue light sensor
  • [http://parts.igem.org/Part:BBa_K592012 BBa_B0015], a terminator

The normal MiniPrep protocol was followed, however the BBa_K608002 and BBa_B0015 Bricks only had 2/3 as much genetic material collected as the other Bricks. (Plasmids collected from 2 Eppendorfs, as opposed to the usual 3)

NanoDrop and Digestions

We are combining...

- a promoter and RBS to the blue light sensor (BBa_K608002 + BBa_K592004)

- a promoter and RBS to the FixJ protein coding region (BBa_K608002 + BBa_K592005)

- the magenta pigment to a terminator (BBa_K592012 + BBa_B0015)

- the yellow pigment to a terminator (BBa_K592010 + BBa_B0015)

First we have to work out the concentrations of each plasmid using a NanoDrop machine.

NanoDrop results

For our promoter and RBS Brick (BBa_K608002) and terminator Brick (BBa_B0015) we have two replicates.

- Promoter and RBS #1 - 29.1 ng/µl

- Promoter and RBS #2 - 25.2ng/µl

- Terminator #1 - 27.6ng/µl

- Terminator #2 - 19.2ng/µl

- Blue light sensor - 38.3ng/µl

- FixJ intermediate - 58.1ng/µl

- Yellow pigment - 45.7ng/µl

- Magenta pigment - 72.0ng/µl

- RFP control - 43.4ng/µl

To get a concentration of 25ng/µl for the restriction digest, we will use...

- Promoter and RBS #1 - 17.18 µl DNA and 25.82 µl distilled water

- Promoter and RBS #2 - 19.84 µl DNA and 23.16 µl distilled water

- Terminator #1 - 18.12 µl DNA and 24.88 µl distilled water

- Terminator #2 - 26.04 µl DNA and 16.96 µl distilled water

- Blue light sensor - 13.05 µl DNA and 24.95 µl distilled water

- FixJ intermediate - 8.61 µl DNA and 34.39 µl distilled water

- Yellow pigment - 10.94 µl DNA and 32.06µl distilled water

- Magenta pigment - 6.94 µl DNA and 36.06 µl distilled water

- RFP control - 11.52 µl DNA and 31.48 µl distilled water

The buffer we are using contains BSA already, so the change from the iGEM Digestion protocol is that 0.5 µl BSA has been replaced with 0.5 µl distilled water, which is included in the values above.

Restriction digest

Our "Part A" Bricks will be BBa_K608002 (promoter and RBS), BBa_K592010 (yellow pigment) and BBa_K592012 (magenta pigment). Our "Part B" Bricks will be BBa_K592004 (blue light sensor), BBa_K592005 (FixJ coding region) and BBa_B0015 (a terminator).

We will also be using pSB1K3 as our plasmid backbone, so our final ligation products should be kanamycin resistant.

Method:

-Labelled PCR tubes with Part A, Part B, pSB1K3 and RFP control (Parts A and B described above)

- Add correct amounts of distilled water and DNA (see "To get a concentration of 25 ng/µl for the restriction digest, we will use...")

- Add 5 µl of 10X FastDigest buffer (which contains BSA) to each tube.

- To the Part A tubes, add 1 µl EcoRI and 1 µl SpeI.

- To the Part B tubes, add 1 µl XbaI and 1 µl PstI.

- To the pSB1K3 tube, add 1 µl EcoRI and 1 µl PstI.

- To the RFP control, add 1 µl EcoRI and 1 µl PstI.

- Mix all by pipetting up and down gently. DO NOT VORTEX. Flick the tubes to collect the liquid at the bottom.

- Incubate in a thermocycler - 37 °C for 30 minutes, then 80 °C for 20 minutes. As a precaution, the final temperature was set at a 4 °C infinite hold, incase we didn't get to the thermocycler immediately when the cycle finished.

Ligation

We need...

- PCR tubes

- distilled water

- T4 DNA ligase reaction buffer

- T4 DNA ligase (MUST be kept in the freezer until last minute)

- pSB1K3 from digest

- Part A from digest

- Part B from digest

- RFP control from digest

Method:

- Label a PCR tube as "New Part" (New Part 1 is promoter and RBS + blue light sensor, New Part 2 is promoter and RBS + FixJ intermediate, New Part 3 is magenta pigment + terminator, New Part 4 is yellow pigment + terminator)

- To these tubes, add 2 µl pSB1K3, 3.3 µl Part A, 3.9 µl Part B, 1.0 µl T4 DNA ligase reaction buffer and 0.5 µl T4 DNA ligase.

- Mix gently by pipetting up and down. DO NOT VORTEX.

- Also label a PCR tubes as ligation control. This will contain 2ul from the RFP control digest, 6.5 µl purite water, 1 µl T4 DNA ligase reaction buffer and 0.5 µl T4 DNA ligase. Mixed gently by pipetting up and down.

- Collect liquid at bottom of all PCR tubes by flicking.

- Incubate in the thermocycler for 30 minutes at 16 °C then 20 minutes at 80 °C. As a precaution, the final temperature was set at a 4 °C infinite hold, incase we didn't get to the thermocycler immediately when the cycle finished.

Our products from ligation were stored overnight at -20 °C to be worked with tomorrow.

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli