Welcome to Benchling!

You're looking at a Notebook entry. Here are some things you can do with it:

Track every step of your experiment

  1. Add new dates for every day your experiment runs.
  2. Capture protocols, observations, and tasks.
  3. Automatically keep a detailed history of your work.
  4. Auto-populate your calendar with Notebook entries.

Insert tables with calculations

Link sequences directly in your entry

Attach images and results

Share data with your collaborators

Create account

Sign up with GoogleI already have an account
Benchling is actively tested against the latest versions of Chrome, Firefox, Safari, and Edge. **It doesn't look like your current browser is supported** - for more information, [click here](/browsers).
Monospace normal
Monospace bold
0 of 0
Advanced Settings
    Creating pBAD24-MoClo (Archived)
    • Notes
    • Metadata
    • Relevant Items
    Editing disabled on archived entry.

     
    Creating pBAD24-MoClo

    Monday, 7/1/2019Aim: create a Golden-Gate compatible pBAD24 plasmid using iGEM prefix & suffix for CDSPrimer sequences for creating pBAD24-MoCloPCR amplification pBAD24GG : Hugues- Daniela- NatachaPCR master mixPCR cycle settingsTuesday, 7/2/2019Wednesday, 7/3/2019pcr.PNG
    Monday, 7/1/2019
    Aim: create a Golden-Gate compatible pBAD24 plasmid using iGEM prefix & suffix for CDS
    ●
    Coding sequences (CDS) cloned into pBAD24-MoClo will be under the transcriptional control of the arabinose-dependent promoter Para. Gene expression will be induced when bacteria harboring the pBAD24-CDS are grown in media containing arabinose. The expression will be repressed when bacteria are grown in media containing glucose (the catabolite repression is ensured by the binding of the CAP protein in the Para promoter of the plasmid).
    ●
    Principle: amplify a DNA fragment with primers 2019GO-3-pBADMoClo-F and 2019GO-4-pBADMoClo-R using plasmid pBAD24GG as a template. Primer 2019GO-3 is phosphylated at the 5'-end. Thus the amplified fragment can be ligate to itself, creating pBAD24-MoClo.
    pBAD24GG
    ●
    PCR amplification pBAD24GG : Hugues- Daniela- Natacha
    Resuspend oligo 2019GO-3-pBADMoClo-F in 557 µL in milliQ water (it’s not at 100 pmol/µL, it’s at 56 pmol/µL)
    Resuspend oligo 2019GO-4-pBADMoClo-R in 617 µL milliq water
    Aliquots of oligos 2019GO-3 and 2019GO-4 were diluted at 1/10 in milliQ water
    Dilution of 2µL pBAD24GG (empty plasmid vector used as template) in 18 µL of milliQ water
    Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):
    This PCR reaction did not work there were no expected fragments on the gel.
    The Tm may have been too high, then this PCR was done again with a lower Tm.
    Tuesday, 7/2/2019
    Repeat PCR amplification from pBAD24GG : Hugues- Daniela- Natacha
    The PCR was runned again in new agarose gels, 1% and 0.7%. There was still no results. Then, the PCR was done again the same manner.
    Wednesday, 7/3/2019
    PCR amplification from pBAD24GG : Hugues-Natacha-Clara
    PCR reaction (from July the 1st) was prepared for loading on agarose gel : 1µL PCR in 6µL of bromophenol blue
    Electrophoresis 30' 100V
    It worked we got a signal at 4.5 kb. It's the correct size of the plasmid. See photo (indicated sizes for marker are in kb).
    pcr.PNG
    Loading...
    Loading...
    Nanodrop quantification of PCR product
    Quantification of plasmid with the nanodrop: 495,657 ng/µL
    Stock 10µL of PCR product at -20°C (BACK UP)
    DpnI digestion
    Add 0,5 µL of DpnI to the rest of plasmid (around 40µL)
    Incubate at 37°C for 1 hour
    20 minutes at 80°C
    Stop on ice
    PCR fragment clean up
    See NucleoSpin Macherey-Nagel protocol
    Nanodrop PCR fragment digested by DpnI
    Quantification of plasmid linearized and digested by DpnI with the nanodrop: 97,421 ng/µL
    Split up pBAD24GG into 2 samples (around 10µL in each)
    Circularization of PCR fragment to create pBAD24-MoClo (empty vector)
    Ligation;
    Add 7µL of water into eppendorf
    +2µL of ligase buffer (10X)
    +1µL ligase (Promega)
    +10µL PCR fragment
    Incubate the mix at 4°C over night
    pBAD24-MoClo
    Transform DH5alpha with ligation product
    The DH5alpha will provide a stock of circularized pBAD24-MoClo ready for Golden Gate cloning.

    Welcome to Benchling!

    Sign in to view data.

    Split Workspace