CHEM Molecular Techniques Flashcards
Explain the principle of Qiagen Symphony
- AUTOMATED nucleic acid EXTRACTION
- chaotropic salt (Proteinase K) lyse cells = cellular components are released
- MAGNETIC SILICA BEADS are used to ADSORB NUCLEIC ACIDS
- a magnetic rod transfers beads to a series of reaction vessels; DNA is washed to remove contaminants
- DNA eluted by a low salt buffer
Concentration of dsDNA when A(260) = 1.000
50 µg/mL
Concentration of ssDNA when A(260) = 1.000
33 µg/mL
Concentration of ssRNA when A(260) = 1.000
40 µg/mL
If a solution of dsDNA has an A(260) of 0.81, what is the concentration of dsDNA ?
(50 µg/mL / 1.000) = ( x / 0.810), so x = 40.5 µg/mL
List the reagents used in endpoint PCR
- DNA polymerase
- dNTPs
- PCR buffer
- Primers
- MgCl2
NOTE: Master Mixes can include all reagents to reduce repetitive measurements
What is the function of Taq polymerase ?
- used in endpoint PCR
- replicates template DNA
- recognizes 3’ end of primer
What can occur if [dNTPs] is not optimal ?
Taq polymerase can be inhibited
What is the function of PCR buffers ?
- maintains optimal pH = 8.3 for Taq polymerase
- provides cofactors (Mg2+) for enzymatic activity
- provides salt for annealing/ hybridization
What can occur if [salt] is too high in endpoint PCR ?
Taq polymerase can be inhibited
Which formula is used to calculate the reagent amounts required for endpoint PCR ?
C1V1 = C2V2
What is endpoint-PCR ? ID the 3 thermocycles
Agarose gel electrophoresis used to amplify nucleic acids:
1. Denaturation (95°C)
2. Annealing/ hybridization (60°C)
3. Elongation (65°C)
Describe the unidirectional workflow requirements of a molecular lab
- amplicons produced in the post-room cannot re-enter the pre-room
- this also applies to PPE and equipment
How many primers are necessary for endpoint PCR ?
- two primers that are complementary to opposite strands of DNA target sequence
- must have similar melting temperature (Tm)
- provides 3’ end for Taq polymerase
What is the function of Mg2+ in endpoint PCR ?
A required cofactor for Taq polymerase
NOTE: but if [Mg2+] is too high, DNA replication can be inhibited
Why is a master mix used in endpoint PCR ?
Reduces error introduced by repeated pipetting of small volumes
How is the master mix prepared in the “clean room” ? What is added outside of the clean room ?
- all reagents EXCEPT the template is added into a single vessel in the clean room = master mix
- DNA template is added outside the clean room
Denaturation step in the PCR thermocyler program
At 92 - 95°C:
- dsDNA breaks to form ssDNA
- 10 to 30 sec, but GC-rich targets can take longer
- hot start Taq DNA polymerase prevents non-specific DNA replication at lower temp. until desired denaturation temp. is reached
Annealing step in PCR thermocyler program
At 50 to 60°C:
- primers bind to complementary sequences on ssDNA template
- takes ~30 sec
- specific temp is determined by melting point (Tm) of primer and ionic concentration of rxn = (Tm - 5°C)
Tm = 4(G+C) + 2(A+T)
How is Tm of PCR primers calculated ?
Tm = 4(G+C) + 2(A+T)
Extension step in PCR thermocyler program
At 72°C:
- template DNA is doubled
- DNA polymerase adds free dNTDs to 3’-end of annealed primer = forms dsDNA
a). one min/ 1kb
b). 30 sec for targets < 1kb
What is RT-PCR ?
- reverse transcription PCR uses an RNA template instead of DNA
- RNA is converted to cDNA (complementary DNA) via reverse transcriptase = first strand synthesis
What is first strand synthesis ?
When RNA is converted to complementary DNA (cDNA) via reverse transcriptase in RT-PCR
Can mRNA be used as the template in RT-PCR ?
yes, RNA sub-fractions like mRNA can be used instead of total RNA
