From 2013.igem.org

Revision as of 10:08, 18 July 2013

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

MiniPrep

We extracted the plasmids for...

- 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)

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_K592004)

- 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 - 29.1ng/ul

- Promoter and RBS #2 - 25.2ng/ul

- Terminator #1 - 27.6ng/ul

- Terminator #2 - 19.2ng/ul

- Blue light sensor - 38.3ng/ul

- FixJ intermediate - 58.1ng/ul

- Yellow pigment - 45.7ng/ul

- Magenta pigment - 72.0ng/ul

- RFP control - 43.4ng/ul

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

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

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

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

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

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

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

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

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

- RFP control - 11.52ul DNA and 31.48ul 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.

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 25ng/ul for the restriction digest, we will use...")

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

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

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

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

- To the RFP control, add 1ul EcoRI and 1ul 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 - 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.

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 2ul pSB1K3, 3.3ul Part A, 3.9ul Part B, 1.0ul T4 DNA ligase reaction buffer and 0.5ul 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, 6.5ul purite water, 1ul T4 DNA ligase reaction buffer and 0.5ul 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 16oC then 20 minutes at 80oC.