Pcb 4522L - Lecture 2

Front 1

----- DNA technology makes manipulating genes possible.

Back 1

Recombinant

the technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism

Front 2

Cloning GFP gene fragment into pL4440

explain

Back 2

Obtain vector (plasmid prep) and gene of interest (PCR or other method)
Prep gene of interest and vector (restriction digest, etc.)
Insert gene into vector (ligation)
Transform ligation products into bacteria
Purify plasmids
Test for insert
Propagate bacteria containing correct clone

Front 3

Where do plasmids come frome?

Back 3

A bacterial plasmid is a strand of DNA inside a bacterium which is independent of the bacterium's chromosomal DNA. Plasmids are capable of replicating on their own, and they can be passed between organisms, an important trait for bacteria, as they use plasmids to transfer genetic information between each other. This ability also becomes important for researchers, who use bacterial plasmids as vectors to insert foreign DNA into DNA they are researching.

Front 4

Recombinant plasmids (aka vectors) contain...

1. origin of ---
2. ---- ---- --- for insert aka -----
it is a recognition site for many -----
3. ----- resistance genes to be used as ----
4. Appropriate ---- for resistance markers and insert

Back 4

replication,
multiple cloning site, polylinker, enzymes
antibiotic, marker
promoters

Front 5

Multiple cloning site

Back 5

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids.[1] Restriction sites within an MCS are typically unique, occurring only once within a given plasmid.

Front 6

What is our vectore and what muct be induced?

Back 6

pL4440, T7 RNA polymerase

Front 7

Induction of T7 RNA polymerase

The T7 phage RNA polymerase gene is controlled by a --- promoter

it is stimulated by ----- , a non hydrolyzable lactose analog

Back 7

lac

IPTG

Front 8

so IPTG --> T7 Polymerase ---> GFP dsRNA made

Back 8

!

Front 9

Lac operon is repressed by the lac repressor which can be inhibited by an induce called

Back 9

IPTG

Front 10

A vector for very large DNA fragments are called..

Back 10

YACs
yeast artificial chromosomes

Front 11

Blue white screen is used in both bacteria and eukaryotic cells. The bacterial lacZ gene encodes a ---- enzyme. When media containing ----- is added, cells expressing the gene convert the ----- to a blue product and can be seen with the naked eye.

Blue colonies are ---- for beta galact
white colonies are ----- for beta galact

What plasmid is used for this?

Back 11

beta-galactosidase,
xgal,
xgal

positive
negative

pUC18

Front 12

The white colonies contain recomb or non recomb DNA molecules?

blue colonies?

Back 12

recombinant

no recombinant

Front 13

Because plasmids can become lost when they reach sizes greater
than 15 kb, scientists developed a system where the E. Coli host
lacZ gene is partially deleted, but activity can be restored by a ~ 30 ---- ---- --- ---- fragment expressed from the plasmid.

Back 13

amino acid lacZ alpha fragment


The lacZ fragment can bind the inactive betagalactosidase that was inactive and activate it to create blue white colonies.

Front 14

Plasmids are uncommon in eukaryotes, thus most eukaryotic vectors are based on DNA or RNA ---- genome

Back 14

viral

Front 15

Delivery of genes by virus is called

Back 15

transduction

Front 16

Three types of restriction endonuclease and what kind of cleavage?

Back 16

Type I - nonspecific cleavage (sorta)
type II: palindromic sequences
Type III: recognition of different sites on each strand

Front 17

SmaI and EcoRI are type --- enzymes


--- produces blunt ends while --- produce sticky ends

Back 17

type 2

SmaI,
EcoRI

Front 18

HindII and KpnI are type ---

--- LEAVE 5' OVERHANGS WHILE --- leaves 3' over hangs

Back 18

HindIII , KpnI

Front 19

DNA frags with blunt ends generated by different enzymes can be ligated together but the ligations must reconstitute the original cut site.

Back 19

!

Front 20

Where does the insert come from?

Back 20

cut it from another vector
order an oligonucleotide - less common
or do a PCR rxn using another vector or chromosomal DNA - most commone

Front 21

PCR - requires gene specific DNA --- and ---

Back 21

primes, dNTPs

Front 22

You anneal primers at the primers' --- minus ---


or you can guess --- for each g/c and --- a/t

Back 22

melting temperature, 5 degrees celsius

4 degrees, 2 degrees

Front 23

for 20 cycles you get how many copies

45?

Back 23

2^20 - 1 million

2^45 - 35 trillion

Front 24

PCR works optimally for ---- bp

Back 24

1000

Front 25

primers typically contain a ---- --- site ~6-10 nucleotides from the end

Back 25

restriction enzyme site

Front 26

Ligations:

cut vector is treated with a ---- to prevent reannealing

the ligation buff contains ATP which is required for the ligase enzyme

Back 26

phosphatase

ATP

Front 27

To make the bacteria competant you...

Back 27

heat shock it

Front 28

Introducing plasmid DNA into a eukaryotic cell is called ---

this is done using ---- which fuse with the cell membrane and spill contents into the cell

Back 28

transfection

liposomes

Front 29

Plasmid DNA can also be introduced via ---- which introduce cracks in the plasma membrane

Back 29

electroporation

Front 30

Explain DNA prep

Back 30

collect bacteria
lyse bacteria
remove protein
and obtain DNA

Front 31

In the old school method: dna was precipitated using --- and ----

newer method is?

Back 31

salt , alcohol

collect on column

Front 32

if you mix a sample with phenol...

dna in aqueous or organic phase?
proteins?

Back 32

dna - aqueous
proteins - organic

Front 33

ratio of vector to insert for ligations?

no more than -- volume of enzyme in restriction digest

Back 33

1:3, more insert than vector

1/10