BME100 s2014:T Group11 L5

From OpenWetWare
Jump to: navigation, search

OUR TEAM

Name: Deven Govin
Name: Hillary Bratlien
Name: Allan Ross
Name: Sharon Li
Name: Osama Wali

Background Information

SYBR Green 1 Dye: The SYBR green 1 dye is a light sensitive liquid that when added to DNA emits a green light and absorbs blue light.

Single-Drop Fluorimeter: The fluorimeter consists of a plastic box with a channel for inserting microscope slides. A switch on the side activates the blue LED that shines through the drop being analyzed.

Fluorescence Technique: This technique is done by combining the SYBR Green Dye with the PCR Samples on the microscope slide. Then a blue light beam is shined through the drop and the dye will emit a different amount of green to

Procedure

Camera Used: Samsung Galaxy S3

Camera Settings:

                ISO: 800
                White Balance: Auto
                Exposure: High
                Saturation: High
                Contrast: Low

Calibration and Solutions Used:

Distance between camera and drop: 6.5 cm

Initial Concentration of 2X Calf Thymus DNA solution (µg /mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I solution (µg/mL)
5 80 80 2.5
2 80 80 1
1 80 80 0.5
0.5 80 80 0.25
0.25 80 80 0.125
0 80 80 0

Placing Samples:

1. micropipette 80 µL of SYBR Green I onto the slide in the middle of the first two rows

2. micropipette 80 µL of Calf Thymus DNA solution into the SYBR Green I

3. move the slide to align the drop with the blue LED

Data Analysis

ImageJ Circle.jpg

PCR Product TUBE LABEL Volume of the DILUTED PCR Product solution (µL) Volume of the SYBR GREEN I Dye solution (µL) INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS ' ' AVERAGE INTDENS VALUE
PCD (30337) 80 80 5947701 6168373 6168373 6094816
P11 80 80 1801358 1548272 1548272 1632634
P12 80 80 1517215 1111331 1340538 1323028
P13 80 80 1642188 1104759 1095043 1280663
PCN (57707) 80 80 1740195 1944156 1560480 1748277
P21 80 80 2689350 1742994 1264839 1899061
P22 80 80 1642865 1407526 1001890 1350760
P23 80 80 1028169 1085183 941678 1018343
PCR Product TUBE LABEL AVERAGE INTDENS VALUE PCR Product Concentration, µg /mL Corrected PCR Product Concentration, µg /mL
PCD (30337) 6094816 0.568964 0.047414
P11 1632634 0.122746 0.010229
P12 1323028 0.091786 0.007649
P13 1280663 0.087549 0.007296
PCN (57707) 1748277 0.13431 0.011193
P21 1899061 0.149389 0.012449
P22 1350760 0.094559 0.00788
P23 1018343 0.061317 0.00511


PCR Results Summary

Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative. Your positive control PCR result was ____ μg/mL Your negative control PCR result was ____ μg/mL

Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed Patient _____ : Patient _____ : Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion. Patient _____ : Patient _____ :