Biochemistry 611 Nucleic Acids 83006

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Biochemistry 611 Nucleic Acids8-30-06
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Supplemental Topics

• Quantitating
• Examples on how to calculate nucleic acids using
  the spectrophotometer
• How to use the 2 4 rule for PCR primers
• Analysis
• DNA Bisulfite genomic sequencing, nested PCR
• RNA RT-PCR, Exon trapping

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Quantitating Nucleic Acids
Example 1 (RNA) You have prepared RNA from a
cell. You measure the RNA using a
spectrophometer and obtain a reading of 68 A260
units. What is the concentration of the RNA?
EQUATION OD260 of sample x 40
concentration of RNA (ug/mL)
68 OD/mL x 40ug/OD 2,720 ug/mL
or 2.72 mg/mL
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Quantitating Nucleic Acids
Example 2 (DNA) You have performed a plasmid
harvest from a bacteria culture. You measure the
DNA using a spectrophometer and obtain a reading
of 25 A260 units. What is the concentration
of the DNA?
EQUATION OD260 of sample x 50
concentration of DNA (ug/mL)
25 OD/mL x 50ug/OD 1,250 ug/mL
or 1.25 mg/mL
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Quantitating Nucleic Acids
Example 3 (Oligonucleotide) You received a tube
of lyophilized primers and need to resuspend them
to 1ug/uL. The tube containing the primer lists
an OD260 of 3.5. What is the concentration of
the DNA? How much water should you add to the
lyophilized primer to bring it to a final
concentration of 1ug/uL?
EQUATION OD260 of sample x 33ug/OD
concentration of DNA
3.5 OD x 33ug/OD 115.5 ug
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Primer Design Calculating the Tm using the
24 rule
TACCTAGGTTGACCATCTACTAA
TACCTAGGTTGACCATCTACTAA 9 GC
TACCTAGGTTGACCATCTACTAA 14 AT
Tm 2C x (AT) 4C x (CG)
Tm 2C x (14) 4C x (9) Tm 28C
36C 64C
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RT-PCR

• Basic Reaction Mixture
• RNA
• dNTPs
• Primers
• 1x Buffer
• Reverse Transcriptase
• RNase Inhibitor
• Thermophilic Polymerase

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Potential Splicing Events in the Exon Trapping
Procedure
exon
exon
exon
exon
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Nested-PCR

• Consists on two consecutive PCR reactions
• The amplified product from the first reaction
  acts as template DNA for the second

1st PCR reaction
2nd PCR reaction
Final PCR product