Unit 3

This was discovered in 1952 in enteric bacteria
-- Circular, double-stranded DNA
-- Can replicate independent of the genome
-- Used in horizontal gene transfer
-- Often contain genes for antibiotic resistance

This is a region within a plasmid where DNA has been engineered to carry many cutting restriction sites. This allows the molecular biologist to use this site as a place to insert fragments of DNA.

This is a plasmid containing an inserted gene, which will carry that gene into the host cell

This is a plasmid containing an inserted gene, which involves combining DNA from multiple sources

This is a protein produced by a recombinant DNA plasmid, typically in a non-native host

This is a feature of a plasmid. It is a DNA sequence that recruits DNA Polymerase for DNA replication

This is a feature of a plasmid. It is the piece of DNA (usually a gene) added to the vector

This is a feature of a plasmid. It is a gene that confers a growth advantage under specific conditions

Selectable Marker v. Screenable Marker

Which is which?

(1) a gene which allows distinguishing bacteria carrying a plasmid that contains an insert versus plasmid that does not contain an insert
GFP allows you to identify which of the surviving bacteria have GFP instead of just an empty plasmid.

(2) a gene that confers a growth advantage under specific conditions
Example: The gene encoding beta-lactamase metabolizes ampicillin and allows growth in the presence of this antibiotic.

More features found on plasmids:

(1) __________ = DNA sequence that recruits RNA polymerase for DNA -> RNA transcription
(2) Coding sequence
(3) __________ = a sequence of DNA that signals for RNA polymerase to detach from the DNA sequence; poly(A) signal in eukaryotes

This is an enzyme that cuts specific DNA sequences (Ex: EcoRI cuts G/AATTC)

-- Common in bacteria (eliminates infectious viral DNA)
-- Some make staggered cuts (sticky ends)
-- Some make straight cuts (blunt ends)
-- Large number are known & commercially available

Remember that __________ _____________ work best by NOT cutting near the end. This will be important when we start designing primers for PCR with restriction enzyme recognition sites. Keep this in mind.

Steps in a Restriction Digest (using EcoRI)

(1) Double-stranded DNA with the EcoRI recognition sequence

(2) Digest with the EcoRI restriction endonuclease (__________ bonds break)

(3) Separation of the DNA strands

Why are restriction endonucleases called ENDOnucleases?

Blunt ends v. Sticky ends

(1) A cut at the center of palindrome

(2) A cut off- center produces cohesive ends. There are 2 types: 3' overhang and 5' overhang

Compatibility of Ends

Compatibility depends on _____ and _______ of the ends!

PRACTICE ENZYME CUTTING IN CAMERA ROLL

OVERVIEW OF CUTS:

(1) All blunt ends are compatible with all other _______ ends


(2) 5′ overhangs are compatible with complementing sequence ends with _' _________


(3) 3′ overhangs are compatible with complementing sequence ends with _' __________

Setting up a Digestion Reaction:

(1) Select a ________ _________ (RE) that will cut DNA at the desired location

(2) Identify specific conditions necessary for the RE to function

(3) Assemble and incubate the restriction digestion reaction

(4) ___ ____________ to confirm that the reaction worked (you have just cut a plasmid into two pieces, how do you make sure that there are 2 pieces of DNA in your sample?)

DO THE DIGESTION REACTION PROBLEM EXAMPLE IN CAMERA ROLL (MESSAGES)

Which of the following plasmids will be used in our course to express CaMPARI in a mammalian system?

a. pHLsec

b. pcDNA3-CaMPARI

c. pET41a-CaMPARI

d. pHLsec-CaMPARI

What data will you get from taking a spectrophotometric reading of your purified plasmid? (Select all that apply.)

a. concentration in ng/μL

b. image of the absorbance curve

c. fluorescence index

d. the A260/A280 ratio

What information can you determine from the results of a spectrophotometric reading? (Select all that apply.)

a. The length of the DNA

b. The concentration of the DNA

c. The shape of the DNA

d. The purity of the DNA

Which of the following methods will we use to separate plasmid DNA from chromosomal DNA? (IN THE SPIN TUBE)

a. NanoDrop/spectrophotometry

b. Alkaline lysis

c. Silica adsorption

d.Restriction digestion

You put a DNA sample on to a spectrophotometer and get a concentration of 384.25 ngL and purity of 1.861 A260/280, what is the DNA yield for 28 uL sample of the DNA? Make sure your answer is in nanograms.

Describe how plasmid DNA is purified by ALKALINE LYSIS and SILICA COLUMN ABSORPTION:
[put these in order along with the story: (PE, P1, EB, N3, P2) ]

(1) First we resuspend the bacterial cells and lyse them in an ________ solution, which raises the pH and causes the chromosomal DNA and plasmid DNA to ________, breaking ________ bonds between complementary base pairs. [__] & [__]

(2) Then the solution is neutralized to a normal pH, causing ______________ DNA and cell debris to ____________ out of the mixture, while plasmid DNA stays soluble. [__]

(3) Then we use centrifugation to remove chromosomal DNA and leave behind the supernatant (plasmid DNA) and the plasmid DNA binds to a _______ membrane in a mini-column.

(4) Then the remaining contaminants are WASHED from the silica membrane. [__]

(5) Finally, the purified plasmid DNA is _______ from the silica membrane and ready for downstream processes [__]

Put in order the following buffers to be used during the column purification of plasmid DNA
(PE, P1, EB, N3, P2)

Describe how spectrophotometry is used to measure the concentration of DNA:

-- Spectrophotometry is based on the principle that molecules such as DNA, RNA, and proteins absorb ______ at specific wavelengths (nm).

-- When a beam of light passes through a solution containing these biological molecules, the amount of light _________ by the molecules is directly proportional to their _____________ in the biological solution

-- The calculated concentration provides an estimate of the amount of ______ present in the sample

-- Overall, spectrophotometry provides a rapid and convenient method for ___________ DNA concentration, which is essential for many molecular biology experiments.

Purity:

For pure DNA, the ideal A260/A280 ratio is _._. Samples with ratios near the ideal are considered to be pure and of good quality.