MCB reagents memorization

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34 Terms

1
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Lysis buffer

detergent disrupting plasma membrane releasing genomic information within, adds salt to solution that will assist in DNA precipitation down the line

2
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RNAse A

degrades RNa to prevent RNA contamination in pure DNA sample

3
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Incubation period for DNA purification

denature cellular proteins for removal from sample

4
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Ice during DNA purification

Prevent refolding proteins, favor hydrophobic aggregation for precipitation out of solution

5
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Precipitation buffer

Precipitate and pellet cellular debris as water soluble materials interact with the buffer ions and hydrophobic protein aggregates precipitate

6
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Addition of isopropanol to DNA purification

ISO is less polar than water- replacing the water molecules that normally surround DNA causing it to precipitate out. Polar salts interact with non polar alcohol and precipitate out of solution

7
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Resuspension of DNA after iso addition

As salts precipitate after iso, resuspension of nucleic acids allows for salt removal

8
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Addition of ethanol to DNA purification

Allows DNA to precipitate while keeping salt suspended

9
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Resuspension in 1x TE

TE is Tris EDTA, where Tris: buffer maintaining pH and EDTA: chelating agent that binds Mg, inactivating leftover nucleases that could degrade DNA

10
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Smearing on genomic DNA gel

Presence of partially degraded RNA fragments

11
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Determining concentration: purpose of 260 nm wavelength?

Ring structures of DNA nucleotides absorb light at this wavelength the most strongly

Problem: RNA has great absorbance here as well

12
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Determining concentration: purpose of 280 nm wavelength? What values indicate high purity?

Aromatic structures in amino acids absorb at this wavelength. A260/A280 value is used to determine purity, where 1.7-2.0 indicate high purity

13
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Purpose of a gel electrophoresis for DNA purification

  • Integrity and length of DNA

  • Searching for RNA bands/smears

  • Unexpected DNA contamination or irregular fragmentation

14
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What about an additional concentration wavelength reading at 320?

Measure turbidity: cloudy/opaque character of solution (Should be clear)

15
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What is an open reading frame?

Sequences of DNA that code for a start codon and at least 100 following amino acids before a stop codon. Theoretically represents a gene that codes for a protein

16
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What size is the fragmentation that occurs in ISO DNA precipitation? Is this a concern?

40-50kb, not a concern, we are working with DNA that is only 2-3 kb long

17
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How do we determine the concentration of our gDNA?

A 260 value X dilution factor X 50 ug/mL = concentration of gDNA

18
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What are the steps for PCR?

Initial cycle: Denature at 95 for 2 minutes

30x cycles: Melt at 95 for 20 sec, Anneal at Tm-5 for 20 sec, Extension at 72 for 3 minutes

Final cycle: Extension at 72 for 3 minutes, store in freezer

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What is the temperature, duration, and purpose of denaturation?

95 for 2 minutes. Separate complex DNA into one linear strand

20
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What is the temperature, duration, and purpose of Melting?

21
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What is the temperature, duration, and purpose of Annealing?

22
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What is the temperature, duration, and purpose of Extension?

23
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What is Herc II?

Pfu mutant

24
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Why do we use Herc II and not Taq polymerase?

Proofreading & affinity

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26
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What is ArchaeMaxx factor? Why do we use it?

Prevent U poisoning from C→U

27
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Why do we use Tris Borate EDTA (TBE) buffer?

28
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How does DNA move thru agarose?

29
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What is the effect of concentration- higher vs lower- of agarose in a gel?

30
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Why do we use ethidium bromide? Why is it a carcinogen?

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Purpose of the positive control

Compare Kanmx to WT length

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Purpose of neg control, no DNA both primers

Do the primers anneal to each other?

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Purpose of neg control,

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