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!

    FIGURE ONE ***** 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

    The PCR program was set up as per the following:

    1. 94˚C for 2 minutes
    2. 94˚C for 30 seconds
    3. 60˚C for 30 seconds
    4. 72˚C for 2 minutes & 30 seconds

      5. (This was repeated for another 25 cycles)

    5. 72˚C for 10 minutes
    6. 4˚C to end.

  • Different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following)
  • Not all possible primer combinations were used due to limitations on the amount of polymerase enzyme available to us
  • The PCR products were run on a 1.2% GelRed stained agarose gel for visualisation (see picture of gel below).

    • Experimental Design – Primer combinations:

    Template DNA Fwd Primer Rvs primer
    DNA1.2 F1 (AT-FWD-1) R1(AT-RVS-1)
    DNA2.2 F1 (AT-FWD-1) R1(AT-RVS-1)
    DNA1.2 F2 (AT-BHO-F) R1(AT-RVS-1)
    DNA2.2 F2 (AT-BHO-F) R1(AT-RVS-1)
    DNA1.2 F3 (AT-FWD-RBS) R1(AT-RVS-1)
    DNA2.2 F3 (AT-FWD-RBS) R1(AT-RVS-1)
    SSH20 (negative control) F1 (AT-FWD-1) R1(AT-RVS-1)

  • A band was seen in lane 6. This lane was using the primer and DNA template combinations of DNA2.2, F3 and R1 primers

    = SUCCESS!!!

  • A PCR product is seen in lane 6 – this is the DNA2.2 template amplified with the F3 (AT-FWD-RBS) and R1 (AT-RVS-1) primers

    = SUCCESS!!

    • FIGURE two ***** Results of the initial PCR ***

    Figure 2. GelRed stained 1.2% agarose gel of initial PCR of A. tumefaciens using various primers.

    In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.2 template with F1 and R1 primer pair.

    In lane 3 is the DNA2.2 template with F1 and R1 primer pair. In lane 4 is the DNA1.2 template with F2 and R1 primer pair. In lane 5 is the DNA2.2 template with the F2 and R1 primer pair. In lanes 6 and 7 there is nothing loaded as the wells were damaged. In lane 8 there is the DNA1.2 template with F3 and R1 primer pair. In lane 9 there is the DNA2.2 template with the F3 and R1 primer pair and in lane 10 there is the ssH2O negative control. A product is seen in lane 9 – this is the DNA2.2 template with the F3 and R1 primers! This means that we have a A. tumefaciens bacteriophytochrome product with a ribosome binding sight inserted.

    30th August 2010

    PCR Optimization

  • Now that we have a product obtained by the initial PCR it is time to optimize the PCR conditions using the successful DNA2.2 PCR product obtained from last week and the original DNA2.2 template
  • The reaction mastermix for the 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

    The PCR program was set up as per the following:

    1. 94˚C for 2 minutes
    2. 94˚C for 30 seconds
    3. 60˚C for 30 seconds
    4. 72˚C for 2 minutes & 30 seconds

      5. (This was repeated for another 25 cycles)

    5. 72˚C for 10 minutes
    6. 4˚C to end.

  • Again, different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following)
  • Now that we don’t have a limited enzyme supply, even more primer combinations can be used!!
  • The PCR products were run on a GelRed stained 2% agarose gel using a 100bp ladder for visualization
  • All primer combinations tested worked with bands visible in each lane! The strange band pattern seen in lane 6 is most probably due to non-specific binding.

    • Experimental Design – Primer combinations:

    Template DNA Fwd Primer Rvs primer
    DNA2.2 F1 (AT-FWD-1) R1(AT-RVS-1)
    PCR product (from DNA2.2) F1 (AT-FWD-1) R1(AT-RVS-1)
    DNA2.2 F2 (AT-BHO-F) R1(AT-RVS-1)
    PCR product (from DNA2.2) F2 (AT-BHO-F) R1(AT-RVS-1)
    DNA2.2 F3 (AT-FWD-RBS) R1(AT-RVS-1)
    PCR product (from DNA2.2) F3 (AT-FWD-RBS) R1(AT-RVS-1)
    DNA2.2 F1 (AT-FWD-1) R2 (AT-AHO-R)
    PCR product (from DNA2.2) F1 (AT-FWD-1) R2 (AT-AHO-R)
    DNA2.2 F2 (AT-BHO-F) R2 (AT-AHO-R)
    PCR product (from DNA2.2) F2 (AT-BHO-F) R2 (AT-AHO-R)
    DNA2.2 F3 (AT-FWD-RBS) R2 (AT-AHO-R)
    PCR product (from DNA2.2) F3 (AT-FWD-RBS) R2 (AT-AHO-R)
    ssH2O (-) control F1 (AT-FWD-1) R1 (AT-RVS-1)

    FIGURE THREE ***** PCR optimization results ***

    Figure 3. GelRed stained 2% agarose gel of optimized PCR of A. tumefaciens bacteriophytochrome using various primers. In lanes 1 and 15 there is a 100bp ladder. In lane 2 is the DNA2.2 sample with F1 and R1 primer pair. In lane 3 there is PCR product template (from DNA2.2 sample) with F1 and R1 primer pair. In lane 4 there is DNA sample with F2 and R1 primer pair. In lane 5 is the PCR product (from DNA2.2 sample) with F2 and R1 primer pair. In lane 6 there is DNA2.2 sample with F3 and R1 primer pair. In lane 7 there is PCR product template (from DNA2.2 sample) with F3 and R1 primer pair. In lane 8 there is DNA2.2 sample with F1 and R2 primer pairs. In lane 9 there is PCR product template (from DNA2.2 sample) with F1 and R2 primer pairs. In lane 10 there is DNA2.2 sample with F2 and R2 primer pairs. In lane 11 there is PCR product (from DNA2.2 sample) with F2 and R2 primer pair. In lane 12 there is DNA2.2 sample with F2 and R2 primer pair. In lane 13 there is PCR product (from DNA2.2 sample) with F3 and R2 primer pair. In lane 14 there is ssH2O negative control with F1 and R1 primer pair. All the lanes worked. There is a weird result in lane 7 which is probably due to non-specific binding. = SUCCESS!

    3rd September 2010

    PCR Optimization (using Gradient PCR)

  • Now that we have the A. tumefaciens bacteriophytochrome product as well as the RBS inserted it is now time for us to amplify the bacteriophytochrome product with the heme oxygenase gene
  • We will be inserting the two genes in two different orientations in the operon
  • We will achieve this using two different primer pairs
    • The first primer pair is F2 (AT-BHO-F) with R3 (AT-BHO-R) [this will insert the bacteriophytchrome gene BEFORE the heme oxygenase gene in the operon]
    • The second primer pair is F4 (AT-AHO-F) with R2 (AT-AHO-R) [this will insert the bacteriophytochrome gene AFTER the heme oxygenase gene in the operon]
  • A technique called gradient PCR will be used here. This PCR includes different annealing temperatures so that the optimum annealing temperature for the primers can be determined.
  • This should also result in reduced non-specific binding that was observed in the previous PCR result
  • The reaction mastermix for the 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

    The PCR program was set up as per the following:

    1. 94˚C for 2 minutes
    2. 94˚C for 30 seconds
    3. 60˚C for 30 seconds
    4. 72˚C for 2 minutes & 30 seconds

      5. (This was repeated for another 25 cycles)

    5. 72˚C for 10 minutes
    6. 4˚C to end.

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