BME100 f2015:Group7 8amL5

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Contents

OUR TEAM

Name: Tristan Loveday
Name: Tristan Loveday
Name: Jeremy Atkinson
Name: Jeremy Atkinson
Name: Kelly Harper
Name: Kelly Harper
Name: Andrew Medina
Name: Andrew Medina
Name: Farah Alyasari
Name: Farah Alyasari
Name: Rafael Lopez
Name: Rafael Lopez


LAB 5 WRITE-UP

PCR Reaction Report

The pipetting went fairly smoothly because we took our time to make sure all the samples were transferred properly. We made sure that we understood the difference between the first and second stop on the pipettor before we started pipetting, and there was no visible difference between the different PCR reaction tubes after we were finished. While we were pipetting, we noticed some water on the sides of the tubes with samples and master mix; we tried to use the pipette tip to move these water bubbles down to the rest of the liquid, with which we were fairly successful. At the end, there was a small amount of liquid left in a couple of the master mix/sample tubes; however, the amount of liquid was very small and hopefully had no impact on our reactions. We kept our labeling scheme the same, as our labels fit on the PCR tubes.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone:Galaxy Note 5
    • Flash:off
    • ISO setting:800
    • White Balance: auto
    • Exposure:highest
    • Saturation:highest
    • Contrast:lowest


Camera set-up

Image:Example328746598723.jpg

  • Distance between the smart phone cradle and drop =6 cm


Placing Samples onto the Fluorimeter

  1. Place a clean slide onto the stand, with the beam of light going across the slide between the first and second row of dots
  2. Using the micropipeter, place 80 µL of CYBR GREEN 1 between the first and second row of dots
  3. Repeat step 2 using 80 µL of the sample, creating a droplet of 160 µL
  4. set up the smartphone and cradle at 6 cm from the droplet
  5. enclose the smartphone cradle and stand in the lightbox
  6. Take a focused picture of the droplet, ensuring that the lightbox is closed to eliminate excess light
  7. repeat step 6 for a total of 3 pictures of the droplet
  8. Remove the droplet using the micropipeter and dispose of into a liquid contamination container
  9. Repeat steps 2-8 between two rows of the slide that have not been used, using a new slide if the current slide has no remaining sections



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA


High: Image:High94687Thymus.jpg Low: Image:low9687Thymus.jpg Zero: Image:zero94687Thymus.jpg Calibrator Mean Values


Image No. Initial Concentration of 2X Calf Thymus DNA solution (µg/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN 1 Dye solution (µL) Final DNA concentration in SYBR Green 1 solution (µg/mL) Area Mean Pixel Value RAW INTDEN of the drop RAW INTDEN of background drop-background mean stdev
10808009409258.5375507906893875461403138460141155168
20808009409254.7435150917744439440647838460141155168
30808009409236.5123435490917955251753538460141155168
10.2580800.1259409240.1363776479914123286235684194865612025
20.2580800.1259409296.2179053251741990831126184194865612025
30.2580800.12594092153.958144862314013891408484284194865612025
10.580800.2594092154.642145488814067681414211313686515740442
20.580800.2594092155.801146596365743601408527613686515740442
30.580800.2594092141.559133196044874481283215613686515740442
1180800.594092150.0741412079857823613542526165564082620299
2180800.594092195.209183675687289918294669165564082620299
3180800.594092192.0431806969923770417831995165564082620299
128080194092175.33716497850140985163568651630275879202
228080194092176.3116589390249831163395591630275879202
328080194092174.25616396106184256162118501630275879202
1580802.594092189.254178343871358851769850217674084127357
2580802.594092188.669177522112159251753628617674084127357
3580802.594092190.491179236591361941778746517674084127357


Calibration curves

Image:Calibration13643245.jpg Image:Calibration2234245345.jpg

Images of Our PCR Negative and Positive Controls

Negative: Image:negative846115control.jpg Positive: Image:positive846115control.jpg

PCR Results: PCR concentrations solved

Image No. Sample No. Area Mean Pixel Value RAW INTDEN of the drop RAW INTDEN of background drop-background mean stdev
17- +94092112.907106236621622521046141098456941152910
27- +94092113.917107186411586151056002698456941152910
37- +9409296.1669048494532848851564698456941152910
17-1.19409298.454926371952734987363708437331639508
27-1.19409287.533823803453494677030888437331639508
37-1.19409297.762919862532608888725378437331639508
17-1.29409296.725910103219741089036229567110577777
27-1.294092110.9651044093948155899593819567110577777
37-1.294092113.2191065299181466298383299567110577777
17-1.394092108.58710217171990971830745363679655252061
27-1.394092109.50910303910713864959004663679655252061
37-1.394092105.4799924751718355920639663679655252061
17- -94092106.6451003442171830493161178809274446639
27- -9409296.731910159862832884732708809274446639
37- -9409299.797939006575162886384378809274446639
17-2.194092100.6639471557750034872152373720111181741
27-2.19409276.4917197149674898652225173720111181741
37-2.19409280.1377540242667983687225973720111181741
17-2.29409280.5757581486664167691731979304103996626
27-2.294092133.004125146471785561233609179304103996626
37-2.29409255.9395263418725598453782079304103996626
17-2.394092199.4751876897630279218466184122243005407461
27-2.394092107.828101457419016739244068122243005407461
37-2.394092103.18197084987458508962648122243005407461


PCR Results: Summary

  • Our positive control PCR result was 3.691388 μg/mL
  • Our negative control PCR result was 1.618549334 μg/mL


Observed results

  • Patient 1 (54502) : 1-1: 0.874663334 μg/mL 1-2: 3.134221334 μg/mL 1-3: -3.26407 μg/mL

The samples for patient 1 appeared very clear. It is impossible to distinguish any green color by simply looking at the images.

  • Patient 2 (84174) : 2-1: -1.255978 μg/mL 2-2: -0.13918 μg/mL 2-3: 8.4486 μg/mL

The samples from 2-1 and 2-2 are both clear. However, the samples from 2-3 appear green.

Conclusions

  • Patient 1 (54502) : All the pictures from patient 1 appeared clear, and two of the samples came out below the negative control value. Every sample was below the positive control value. For these reasons, the patient 1 sample failed to replicate during PCR and is therefore negative.
  • Patient 2 (84174) : Our data for patient 2 appears to be flawed based on the fact that two of the samples came out below the negative control, while one came out far above the positive control. This correlates with the visual appearance of our pictures. Because sample 2-3 had such a high DNA concentration value, it appears to be an outlier that may be the result of contamination. Based on samples 2-1 and 2-2, patient 2's DNA also failed to replicate, and is therefore negative.


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