BME103 s2013:T900 Group2

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(Research and Development)
(LAB 1 WRITE-UP)
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'''Thermal Cycler Program'''<br>
'''Thermal Cycler Program'''<br>
 +
The set-up used for the program is as follows<br>
 +
Stage one: 1 cycle, 95 degrees Celsius for 3 minutes<br>
 +
Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds<br>
 +
Stage three: 72 degrees Celsius for 3 minutes <br>
 +
Final Hold: 4 degrees Celsius
'''DNA Sample Set-up'''<br>
'''DNA Sample Set-up'''<br>
Line 53: Line 58:
{| {{table}}
{| {{table}}
|-
|-
-
| row 1 cell 1 || row 1 cell 2 || row 1 cell 3 || row 1 cell 4
+
| Positive control:<br>cancer DNA template<br> Tube label: 1 || Patient 1<br>ID: 91862<br> Replicate 1<br> Tube label: 2 || Patient 1<br> ID: 91862<br> Replicate 2<br> Tube label: 3 || Patient 1<br> ID: 91862<br> Replicate 3<br> Tube label: 4
|-
|-
-
| row 2 cell 1 || row 2 cell 2 || row 2 cell 3 || row 2 cell 4
+
| Negative Control: no<br> DNA template<br> Tube label: 5 || Patient 2<br>ID: 28235<br> Replicate 1<br> Tube label:6 || Patient 2 <br> ID: 28235<br> Replicate 2<br> Tube label: 7 || Patient 2<br> ID: 28235<br> Replicate 3<br> Tube label: =)
|}
|}
'''DNA Sample Set-up Procedure'''
'''DNA Sample Set-up Procedure'''
-
# Step 1
+
# Step 1: First gather the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes)
-
# Step 2
+
# Step 2: Set the pipette to 50 microliters
-
# Step 3...
+
# Step 3: Place the transfer pipette onto the pipette to prevent cross contamination (never re-use).
 +
# Step 4: Use the pipette to transfer 50 microliters of each tube in the PCR reaction mix and transfer accordingly to the DNA sample tubes corresponding to the labels.
'''PCR Reaction Mix'''
'''PCR Reaction Mix'''
-
* What is in the PCR reaction mix?
+
* The PCR reaction mix contains 8 tubes of 50 microliter samples that contain a mix of Taq DNA polymerase, MgCl2, dNTP's, forward primers, and reverse primers.
'''DNA/ primer mix'''
'''DNA/ primer mix'''
-
* What is in the DNA/ primer mix?
+
* The DNA/primer mix consists of samples of DNA of various patients.

Revision as of 00:57, 26 March 2013

BME 103 Spring 2013 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Contents

OUR TEAM

Andy SonProtocol Planner
Andy Son
Protocol Planner
William Scott R&D Scientist
William Scott
R&D Scientist
Joe Sasnone R&D Scientist
Joe Sasnone
R&D Scientist
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
(Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is)


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer)

When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)


Test Run

(Write the date you first tested Open PCR and your experience(s) with the machine)




Protocols

Thermal Cycler Program
The set-up used for the program is as follows
Stage one: 1 cycle, 95 degrees Celsius for 3 minutes
Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds
Stage three: 72 degrees Celsius for 3 minutes
Final Hold: 4 degrees Celsius

DNA Sample Set-up

Positive control:
cancer DNA template
Tube label: 1
Patient 1
ID: 91862
Replicate 1
Tube label: 2
Patient 1
ID: 91862
Replicate 2
Tube label: 3
Patient 1
ID: 91862
Replicate 3
Tube label: 4
Negative Control: no
DNA template
Tube label: 5
Patient 2
ID: 28235
Replicate 1
Tube label:6
Patient 2
ID: 28235
Replicate 2
Tube label: 7
Patient 2
ID: 28235
Replicate 3
Tube label: =)

DNA Sample Set-up Procedure

  1. Step 1: First gather the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes)
  2. Step 2: Set the pipette to 50 microliters
  3. Step 3: Place the transfer pipette onto the pipette to prevent cross contamination (never re-use).
  4. Step 4: Use the pipette to transfer 50 microliters of each tube in the PCR reaction mix and transfer accordingly to the DNA sample tubes corresponding to the labels.

PCR Reaction Mix

  • The PCR reaction mix contains 8 tubes of 50 microliter samples that contain a mix of Taq DNA polymerase, MgCl2, dNTP's, forward primers, and reverse primers.

DNA/ primer mix

  • The DNA/primer mix consists of samples of DNA of various patients.






Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase Chain Reaction (PCR) is a scientific method that utilizes DNA Polymerase to create a complimentary base strand from a template strand of DNA. Triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. One major issue with DNA polymerase is that DNA strands are anti parallel, and polymerase is only able to add nucleotides to the free 3'OH group hence it can only build new strands in the 5'-3' direction (Sadava 279). Therefore, to correct for this issue the presence of a primer is required so that polymerase can proceed with directing the new nucleotides in place. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify(PCR 1). When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons).

The SNP included in the amplicon for this experiment was denoted as rs17879961. This polymorphism is a variant of the CHEK2 gene which if present in a person's genome may increase the risk of developing breast cancer (Brennan et al 1795). This SNP is signified by a single base change from a Thymine (T) to a Cytosine (C) located on chromosome 22.

(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)

Sources:

Brennan, Paul et al. Uncommon CHEK2 Mis-sense Variant and Reduced Risk of Tobacco-related Cancers: Case-control Study. Rep. 15th ed. Vol. 16. Oxford: Advance Access, 2007. Human Molecular Genetics. Oxford University Press, 21 May 2007. Web. 25 Mar. 2013. PDF. <http://hmg.oxfordjournals.org/content/16/15/1794.full.pdf>.

"PCR Introduction." NCBI. U.S. National Library of Medicine, n.d. Web. 25 Mar. 2013. <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml>.

Sadava, David E. Life: The Science of Biology. Sunderland, MA: Sinauer Associates, 2011. Print.






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