1. Oligose - Primers Qiagen Dneasy Polymerase Chain Reaction What Does it all Mean? Maria Brown October 22, 1009

2. Oligose (Stock) Integrated DNA Technologies
Sequence: 12Sai
5’ - AAA CTA GGA TTA ACC CTA TTA T-3”
Amount in solid form (33.9 nMoles)
Sequence 12Sbi
5’ - AAA AGC GAC GGG CGA TCT GT - 3”
Amount in solid form (30.8 nMoles)

3. Oligose Dilution (Stock)
Sai Sbi
33.9 nMoles nMoles
To convert to picomoles /uL
ADD 1000 uL
Sai Sbi
33.9 picomoles/uL picomoles/uL
GUARD FOR LIFE!

4. “Working Stock” Rule: what you want x vol. want what you have
To make 12Sai working stock:
15 pmol/uL x 200 uL = uL (12Sai Primer)
33.9 pmol/uL
Want 200 uL:
200 uL – 88.49uL = uL dH2O
88.49 ul 12Sai uL dH2O = “Working Stock”

5. Primer Master Mix To Make a Master Mix for 50 Rxn’s:
Want 22.5 uL per Rxn
50Rxn’s x 22.5 uL = 1125 uL (total volume)
Need: 20uL Sai + 20uL of Sbi
1125 uL – 40 uL Sai/Sbi = 1085 dH2O

6. Extracting DNA (mtDNA)
Best results are obtained with fresh material or material that has been frozen.
Cut and mash a tissue sample. Suitable samples may be as little as 100 cells to as much as 5 million cells.

7. Lysis – Breaking Cells & DNA Purification
Buffers ATL (lysis buffer)-
breaks the cells open
Proteinase K - used to digest (remove) protein that might degrade the DNA or RNA during purification.
Samples must be incubated at 57C for 1 hour before adding AL (additional lysis buffer)and ETOH.

8. Precipitating the DNA
This is done when pure ETOH (96 – 100%) is added. DO NOT use denatured alcohol.
DNA is insoluable in ETOH and it will aggregate together forming a “pellet” after centrifugation.

9. Washing the DNA
Washing the DNA is accomplished by adding Buffers AW1 and AW2 followed by centrifugation.

10. Elution (Removal/Separation Stage)
Purified DNA is eluted from the Dneasy Mini spin filter column with buffer AE. This can be done in one or two successive steps.
Centrifugation of the tube with the DNA embedded in the filter will pull the elution buffer through the matrix and the collection tube.
Buffer AE (10mMTris-Cl, 0.5 mM EDTA, pH 9.0) for optimal DNA yield

11. PCR Ready Add 22.5 uL of Primer Master Mix
Add 2.5 uL of Extracted/Purified/Eluted DNA = (PCR-Ready)

12. Ploymerase Chain Reaction (PCR)
a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.
Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification.
As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.

13. Thermocycler – “BUGS” Settings: 90 Minutes 32 cycles of:
94C denaturing for 30 seconds
52C primer annealing for 30 seconds
72C primer extension for 40 seconds
* Can set to run overnight and run Gel next day

14. Gel Electrophoresis 2% Agarose Gels
2 Log DNA Ladder (New England Biolabs)

15. PCR Results E. Berenice Primers + C 40T 41L 42T 2 Log Ladder 40 L 42L

16. Scientists for Better PCR

17. DNA Density Tests – NanoDrop Spectrophotometer

18. Sequencing mtDNA at BNL

19. Mitochondrial DNA Sequencing Analysis

20. Pylogenetic Tree

21. Clustal W Alignment

22. Biotechnology Applications
Medicine
Agriculture
Biofuels
Conservation Biology
Evolution
Genetic Engineering
Gene Therapy
Disease Management
Other?

23. Analyzing results using online analytical tools
Next Time
Analyzing results using online analytical tools