Lecture 6

Question 1

What is spectrophotometer?

Answer 1

Measures the fraction of light that is able to pass through a solution reads the amount of light that is absorbed by cells

Question 2

True or false: The amount of light absorbed (A) is NOT proportional to the concentration of cells in the culture (c)

Answer 2

False
-They are directly proportional
-the more concentrated a sample = the more absorbance

Question 3

What is the Beer-Lambert Law?

Answer 3

A = (a)(lambda)(b)(c)

Question 4

DNA concentration is measured as absorbance at?

Answer 4

260 nm
-DNA, RNA, nucleic acid, ATP are all measured at this absorbance

Question 5

What is measured at 280 nm?

Answer 5

Proteins
-aromatic amino acids

Question 6

What do absorbance A280, A320, and A230 check for?

Answer 6

Contaminants

Question 7

What is the range for A260/A280

Answer 7

1.7-2
DNA/PROTEIN RATIO

Question 8

What does the Nanodrop tell us?

Answer 8

Tell us the concentration in nanograms/microliter and how pure or contaminated the DNA sample is

Question 9

What does the Qubit tells us and how does it work?

Answer 9

The amount of double stranded DNA only (intact DNA) and concentration
-DNA is treated w/ fluorescent dye–>the dye binds to dsDNA–>DNA will glow if it is dsDNA
-colors can mean bases etc

Question 10

Why do we do sequencing?

Answer 10

To know the number and order of ATCG. in DNA

Question 11

What is Shotgun Genome Sequencing?

Answer 11

Start w/ a complete genome copy
-DNA becomes fragmented by enzymes, sonication, or hydroshearing
-Each piece of DNA is cloned & put into a plasmid

Question 12

What is Sanger Sequencing?

Answer 12

Similar to PCR except
-1 primer
-Make ssDNA pieces
-NOT exponential
-2 kinds of nucleotides
-uses Dideoxy nucleotides–>emit fluorescence

Question 13

Explain dideoxynucleotides (ddNTPs) in Sanger Sequencing

Answer 13

When incorporated it causes the termination of primer chain extension
-No 3’ OH group so no more nucleotides can be added

Question 14

Sanger Sequencing steps

Answer 14

1. PCR
2. Electrophoresis
3. Sequence determination

Question 15

What are limitations to Sanger sequencing?

Answer 15

-Must have 1 colony picked for every 2 reactions
-Must do 1 DNA. prep for every 2 reactions
-Must have 1 PCR tube for each reaction
-Must have 1 gel lane for each reaction
This is why it took so long to sequence 3 mill bp

Question 16

How long are Sanger sequences

Answer 16

800-1000 bp

Question 17

How does Next Generation Sequencing work?

Answer 17

Sequencing by synthesis
-DNA is sequenced while it is amplified

Question 18

How big is P fluorescens genome?

Answer 18

6.7 million bp

Question 19

True or false: genome does not need to be cut for sequencing

Answer 19

False
-It must be cut before sequencing

Question 20

What are adapters?

Answer 20

Pieces of KNOWN DNA are added to each fragmented DNA piece

Question 21

What is Tagmentation?

Answer 21

Tagging & Amplification

Question 22

How does DNA get cut and add specific sequences at each end?

Answer 22

Transposome complex contains transposase that cuts the DNA & adds specific sequences at each end

Question 23

What size fragments do transposome generate?

Answer 23

300 bp

Question 24

Why do we need PCR?

Answer 24

It is used to add indexes/barcodesto adapters

Question 25

True or false: PCR needs primers

Answer 25

True

Question 26

Where are indexes/barcodes found?

Answer 26

In the primers

Question 27

Next Gen Sequencing order?

Answer 27

1. Tagmentation (tagging + amplification)
2. Transposome step
3. Index/barcodes addition to amplify the tagged fragments

Question 28

Sequencing by synthesis overview

Answer 28

1. start with genomic DNA
2. Parallel sequencing
3. Alignment
4. Sequencing

Question 29

Next gen sequencing overview

Answer 29

1. Barcodes are added to each fragment (tagmentation)
2. Library fragments are sequenced in parallel
3. Barcodes are used to differentiate reads from each sample
4. Each sets of reads is aligned