Team:Macquarie Australia/Notebook

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PROJECT LAB BOOK

Welcome to the Macquarie University project lab book page!

Here you will find a day-by-day account of our triumphs and failures.

A day-by-day progress for Agrobacterium Tumefaciens Bacteriophytochrome

20th August 2010

Genomic DNA extraction

  • A. tumefaciens genomic DNA extracted using the Invitrogen PureLinkTM Genomic DNA Purification kit as per the manufacturer’s protocols.

  • The extractions were run on a GelRed stained 1% agarose gel and photo taken for visualization (see below).

  • A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA.

  • The extraction was successful for all A. tumefaciens cell lysate samples (labeled DNA1.1, DNA1.2, DNA2 (See figure below)

    • A. tumefaciens genomic DNA extraction agarose results:

    All four DNA samples show a smear of gDNA. Because the samples were treated with RNase there is no band indicative of RNA visible = SUCCESS!

    ***** Results of A. tumefaciens genomic DNA extraction ***

    Figure 1. GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.

    In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.1 flow through, lane 3 is the DNA2.2 flow through, lane 4 is the DNA2.1 flow through and lane 4 in the DNA2.2 flowthrough. All four samples show a smear that is indicative of genomic DNA. The extraction has been successful.

    Nanodrop absorbance readings:

    Genomic DNA sample 260/280 OD ratio Concentration (ng/mL)
    DNA1.1 1.88 351.5
    DNA1.2 1.98 203.1
    DNA2.1 2.14 738.9
    DNA2.2 2.16 709.7

    Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination

    = SUCCESS!

    27th August 2010

    Primer design

  • Various primers were designed manually and using Primer 3 Software package for PCR amplification.

  • The primers were ordered and supplied through XXXXXXXXXXXXXXXXX.

  • There was an array of various primers ordered for amplification of different products. The details of the primers are described below.

    • Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene:

    Primer name Primer Sequence
    F1 (AT-FWD-1) 5’-ATG AGT TCA CAT ACG CCG-3’
    R1 (AT-RVS-1) 5’-TCA GGC AAT TTT TTC CTC-3’

    Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon BEFORE the HO gene:

    Primer name Primer Sequence
    F2 (AT-BHO-F) 5’- AAG GAG ATA TAC ATA TGA TGA GTT CAC ATA CGC CG – 3’
    R3 (AT-BHO-R) 5’- AAG TTG ACA CTC ATA TGA GCC CTC CTT TCA GGC – 3’

    Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene:

    Primer name Primer Sequence
    F1 (AT-FWD-1) 5’-ATG AGT TCA CAT ACG CCG-3’
    R1 (AT-RVS-1) 5’-TCA GGC AAT TTT TTC CTC-3’

    Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon AFTER the HO gene in the operon:

    Primer name Primer Sequence
    F4 (AT-AHO-F) 5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG – 3’
    R2 (AT-AHO-R) 5’- GTT AGC CGG ATC CTC AGG CAA TTT TTT CCT – 3’

    Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon AFTER the HO gene as well as the addition of a ribosome binding site or Shine Delgano sequence:

    Primer name Primer Sequence
    F3 (AT-FWD-RBS) 5’- AGG AGG GCT ATG AGT TCA CAT ACG CCG -3’

    Initial PCR

  • The reaction mastermix for the initial PCR was set up as per the following recipe (per sample):
    • Mastermix: Amount per sample (ul)
    Clean H2O 13.75
    10x Buffer 2.00
    Polymerase enzyme 0.25
    dNTP 1.00
    Fwd primer 1.00
    Rvs primer 1.00
    Genomic DNA 1.00
    Total 20.00

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