lab and listening Flashcards
Concisely describe how to safely dispose the agar plates
Place in the plastic bag and autoclave
Heat melts the plastic agar plate
can be discarded
How to place the agar plate for incubation
upside down so that condensation doesn’t form on the side with the medium
37 degrees C and 5% CO2 atmosphere
Agar plates
petri dishes with an agar medium on it that allows for the growth of microorganisms
can have LB or antibiotics on it as well
LB
a broth created to promote bacterial growth
Ampicillin
an antibiotic that can be on the plate to help with the creation of a specific bacterial colony
antibiotic resistance
Permissive plate
allows the growth of any bacterial colony
Restrictive plate
allows growth of specific colonies based on antibiotic resistance
Indicator plate
shows colonies based on color
List the major procedures to making agar plates
Add 500 mL of distilled water into a 1 L flask
Insert LB agar tablets
cover with aluminum foil
autoclave
50 mg Amp and dissolve in 1 mL distilled water
LB flask cools to 55 degree C, add Amp mixture
Pour the liquid onto each plate (don’t open the lids all of the way) - about 5 mm
Swirl the plates in a circular motion, let cool for 20 min, incubate upside down
We have 60 uL digested DNA solution and 4X blue dye DNA loading buffer. In order to run DNA gel, we need to add a certain volume of 4X blue dye DNA loading buffer into DNA solution to obtain a final mixed solution with IX blue dye. Please calculate how much volume of 4X blue dye DNA loading buffer which you need to take and add it into DNA solution
60 + x = 4X
60 = 3X
20 = X
ゲ—ム
game
アリバイト
part time job
バイト
part time job
かいもの
shopping
クラス
class
いぬ
dog
ねこ
cat
ひと
person
こども
child
あなた
you
いす
chair
つくえ
desk
しゃしん
picture
はな
flower
レポ—ト
term paper
ごはん
rice
パン
bread
おてら
temple
こうえん
park
ス—パ—
supermarket
バスてい
bus stop
びょういん
hospital
ホテル
hotel
ほにゃ
bookstore
まち
town, city
レストラン
restaurant
geemu
game
aribaito
part time job
baito
part time job
kaimono
shopping
kurasu
class
inu
dog
neko
cat
hito
person
kodomo
child
anata
you
isu
chair
tsukue
desk
shashin
picture
hana
flower
repooto
term paper
gohan
rice, meal
pan
bread
otera
temple
kouen
park
suupaa
supermarket
basutei
bus stop
byouin
hospital
hoteru
hotel
honnya
bookstore
machi
town, city
resutoran
restaurant
We have 50 uL digested DNA solution and 5X blue dye DNA loading buffer. In order to run DNA gel, we need to add a certain volume of 5X blue dye DNA loading buffer into DNA solution to obtain a final mixed solution with 1X blue dye. Please calculate how much volume of 5X blue dye DNA loading buffer which you need to take and add it into DNA solution
50 + X = 5X
50 = 4x
12.5 = x
What do plasmids mean
Double-stranded
circular DNA
in bacteria
used for transformations of foreign DNA into bacterial cells
covalently closed
Are plasmids chromosomal molecules
No
extrachromosomal DNA that’s self-replicating
Vectors
used to carry exogenous DNA from somewhere else and insert it into a host cell
Plasmid vector
vectors where a plasmid is used to carry the exogenous DNA, so it’s transformed into recombinant DNA and introduced back into the cell
Competent cells
capable of taking exogenous DNA and have it inserted into their cells via transformation
What is the purpose to use competent cells
capable of taking exogenous DNA and have it inserted into their cells through a transformation
higher efficiency
used to propagate and maintain cloned DNA in plasmids
Transformation
exogenous DNA is taken into the new bacteria
replaces a section on the plasmid (Lac Z)
Major steps of a transformation
thaw competent cells
mix 2 uL sample and 50 uL competent cells
keep on ice for 30 min
heat shock 35 sec
ice 2 min
add LB
incubate on shaker at 37 degree C for 1 hour
plate on agar plate using sterile beads
incubate 37 degree C overnight
During transformation, which heat shock temperature is used
42 degree C
Why are heat shock time and temperature crucial
you don’t want to destroy your DNA or your cell
you want to heat it enough so that the DNA can enter the cell, but not too long that your bacteria cells begin to die
heat shock allows the DNA to enter through the membrane
What is the purpose to use the appropriate antibiotic during the transformation procedure
antibiotic resistance
transformed bacteria will be given an antibiotic resistance gene, so all non-transformed bacteria will die on the agar plate and the transformed bacteria will grow
Amp plate for Amp resistance
きのう
yesterday
…じかん
hour counter (duration)
いちじかん
for one hour
せんしゅう
last week
とき (の)
when…, at the time of…
げつようび
Monday
かようび
Tuesday
すいようび
Wednesday
もくようび
Thursday
きにょうい
Friday
あう (に)
to meet, to see (a person)
ある (に, が)
there is…
かう
to buy
かく
to write
とる
to take (a picture)
まつ
to wait
わかる (が)
to understand
いる (に, が)
there is… (living thing)
ぐらい
about (approximate measurement)
ごめんなさい
I’m sorry
それから
and then
だから
so, therefore
たくさん
many, a lot
と
together with (a person), and
どうして
why
ひとりで
alone
もし もし
hello? (on the phone)
kinou
yesterday
jikan
hour counter (duration)
ichijikan
for one hour
senshuu
last week
toki (no)
when…, at the time of…
getsuyoubi
monday
kayoubi
tuesday
suiyoubi
wednesday
mokuyoubi
thursday
kinyoubi
friday
au (ni)
to meet, to see (a person)
aru (ni, ga)
there is…
kau
to buy
kaku
to write
toru
to take (a picture)
matsu
to wait
wakaru (ga)
to understand
iru (ni, ga)
there is… (living thing)
gurai
about (approximate measurement)
gomennasai
i’m sorry
sorekara
and then
dakara
so, therefore
takusan
many, a lot
to
together with (a person), and
doushite
why
hitoride
alone
moshi moshi
hello? (on the phone)
What do restriction endonucleases or restriction enzymes mean
enzymes in bacteria created as a defense mechanism to digest foreign DNA
breaks DNA at a recognition sequence with 2 breaks (1 through backbone, 1 through double helix)
Recognition sequence
DNA-binding protein motif where restriction enzymes cut the DNA
blunt end
sticky end
overhanging sing-strand
how to set up a restriction endonuclease reaction
enzyme on ice
spin 30 sec
add buffer, BSE, and enzyme to DNA and water
spin 30 sec to mix
37 degree C water bath for 3 hours (or overnight)
Alkaline phosphatase
antarctic phosphatase
difference between alkaline and Antarctic phosphatase
How to use CIP or Antarctic phosphatase
use NE buffer to dissolve DNA
add vector DNA and CIP
continue on with the steps
centrifuge 30 sec
incubate 37 degree C for 1 hour
purify using a gel (cut out bands, dissolve ,etc)
How to prepare for 1 mL DNA solution in dH2O by diluting your DNA sample with a 1:250 dilution factor
1:250
2:500
3:750
4:1000
4:996
みぎ
right
ひだり
left
まえ
front
うしろ
back
なか
inside
うえ
on
した
under
ちかく
near, nearby
となり
next
あいだ
between
migi
right
hidari
left
mae
front
ushiro
back
naka
inside
ue
on
shita
under
chikaku
near, nearby
tonari
next
aida
between
List major steps in QIAprep miniprep procedure
Add LB and centrifuge in falcon tube
discard supernatent
resuspend pellet in p1
add p2 and mix by inversion
add n3 and mix by inversion
put in column tube and centrifuge
discard supernatent
add PE. centrifuge. discard supernatent
spin empty
change column to new microcentrifuge tube
add elution buffer as close to the filter as possible without touching it
let sit for 1 min. centrifuge. discard column
What is the purpose of LyseBlue
color indicator that shows optimum buffer mixing
prevents errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris
at is the purpose of RNaseA
removes RNA from genomic DNA
function of Buffer P2
lysis buffer
function of Buffer N3
creates all of the right binding conditions to the membrane
At what wavelength does the maximum absorption of UV light occur for DNA
260 nm
At what wavelength does the maximum absorption of UV light occur for proteins
280 nm
What is a ratio of OD 260 / OD 280 that is less than 1.8 indicative of in relation to DNA
protein contamination in your sample DNA
Do not allow the lysis reaction to proceed for more than 5 min after adding buffer p2. why
if you go longer than 5 min, then you’ll have more chromosomal DNA and it’ll begin to denature the plasmid
keeping it below 5 min allows for the release of plasmid DNA from the cell
how to prepare for 1 mL dna solution dH2O by dilution your sample with 1:200 dilution
1 uL for 200 uL
2 uL for 400 uL
3 uL for 600 uL
4 uL for 800 uL
5 uL for 1000 uL
995 uL of dH2O and 5 uL of DNA
What is the function of QIA spin column
separates DNA we want from all of the waste and other parts that we don’t want (cell debris, RNA, proteins, etc)
PB helps it bind to the filter
When spun, everything we don’t want goes through the filter and DNA binds to the filter
PE is the wash buffer and it cleans off waste from DNA
Elution buffer brings the DNA through the filter
ついたち
1st
ふつか
2nd
みっか
3rd
よっか
4th
いつか
5th
むいか
6th
tsutachi
1st
futsuka
2nd
mikka
3rd
yokka
4th
itsuka
5th
muika
6th
なのか
7th
ようか
8th
ここのか
9th
とおか
10th
nanoka
7th
youka
8th
kokonoka
9th
tooka
10th
DNA gel electrophoresis
when you use a gel made from agarose to separate DNA molecules using an electric current
How does DNA gel electrophoresis work
agarose gel made from powder agarose and liquid buffer (TAE or TBE)
placed in the gel chamber
once solid, placed in the gel machine with the same liquid buffer (which is ion-rich)
because phosphate groups on DNA have a net negative charge, DNA migrates to the anode (positive pole)
pores in the agarose gel that are responsible for the way different sizes of DNA migrate through
What is the function of DNA ladders
goes in the 1st or last well
contains different components of known different sizes
when you run a gel, it looks like a ladder and can be used to compare your samples to
Ethidium bromide function
DNA stain that binds to DNA and absorbs UV light
gives off orange visible light when placed on UV transilluminator
Which DNA fragments run fast and why
smaller fragments
pores in the gel allow smaller fragments to get through easier than larger ones
DNA loading buffer
makes DNA denser than the electrophoresis buffer
can also contain dyes so that you can see the DNA after running the gel
it will make the sample sit at the bottom of the wells
UV transiluminator
gives off UV light on the gel after running so that you can see your fluorescently-dyed DNA
In gel electrophoresis, DNA fragments migrate to…
the anode (red) because the phosphate groups are negative on the DNA
function of TAE buffer
mixture of TRIS, EDTA, and glacial acetic acid
TRIS - buffer that works between a pH of 7-9
EDTA - metal chelator that binds to metal ions that are a cofactor for DNase
Glacial acetic acid - provides the proper ion concentration
TAE should be used for separating large fragments, cloning, and DNA extraction from a gel
allow nucleic acids to move through the agarose matrix
Function of EDTA in TAE buffer
metal chelator that binds to metal ions like magnesium, which are required cofactor for DNase activity
たべもの
food
のみもの
drink
くだもの
fruit
やすみ
holiday, day off, absence
りょこう
travel
うみ
sea
サ—フイ(little)ン
surfing
おみやげ
souvenir
バス
bus
てんき
weather
しゅくだい
homework
テスト
test
たんじょうび
birthday
へや
room
ぼく
I (used by men)
エルサイズ
size L
あたらしい
new
ふるい
old
あつい
hot
さむい
cold (weather)
いそがしい
busy
おおきい
large
ちいさい
small
おもしろい
interesting, funny
つまらない
boring
やさしい
easy (problem), kind (person)
むずかしい
difficult
かっこいい
good-looking
こわい
frightening
tabemono
food
nomimono
drink
kudamono
fruit
yasumi
holiday, day off, absence
ryokou
travel
umi
sea
saafin
surfing
omiyage
souvenir
basu
bus
tenki
weather
shukudai
homework
tesuto
test
tanjoubi
birthday
heya
room
boku
I (used by men)
erusaizu
size L
atarashii
new
furui
old
atsui
hot
samui
cold (weather)
isogashii
busy
ookii
large
chiisai
small
omoshiroi
interesting, funny
tsumaranai
boring
yasashii
easy (problem), kind (person)
muzukashii
difficult
kakkouii
good-looking
kowai
frightening
What does PCR mean
technique to amplify a specific segment of DNA into many copies
major pcr steps
denature - hot temp to separate DNA strands
annealing - lower temp when primers attach to ssDNA
elongation - when DNA polymerase creates 2nd strand
dna polymerase
enzymes that helps amplify dna
uses ssdna as a template to make complementary strand
needs a primer
what are dna primers and what do they do
made of RNA and DNA bases
first 2 bases always RNA
synthesized by primase
anneals to the ssDNA to create a 3’ OH group for DNA polymerase to begin working
taq polymerase
thermostable DNA polymerase
lacks 3’ to 5’ exonuclease proofreading activity
たのしい
fun
やすい
cheap
tanoshii
fun
yasui
cheap
すき (な)
to like
きらい (な)
to dislike
suki (na)
to like
kirai (na)
to dislike
だいすき (な)
to love
だいきらい (な)
to haite
きれい (な)
beautiful, clean
げんき (な)
healthy, energetic
しずか (な)
quiet
にぎやか (な)
lively
ひま (な)
not busy
daisuki (na)
to love
daikirai (na)
to hate
kirei (na)
beautiful, clean
genki (na)
healthy, energetic
shizuka (na)
quiet
nigiyaka (na)
lively
hima (na)
not busy
およぐ
to swim
きく (に)
to ask
のる (に)
to ride, to board
やる
to do, to perform
でかける
to go out
oyogu
to swim
kiku (ni)
to ask
noru (ni)
to ride, to board
yaru
to do, to perform
dekakeru
to go out
いっしょに
together
すごく
extremely
だいじょうぶ
it’s okay
not to worry
everything is under control
とても
very
どんな
what kind of…
まい
counter for flat objects
isshoni
together
sugoku
extremely
daijoubu
it’s okay
everything is under control
not to worry
totemo
very
donna
what kind of…
mai
counter for flat objects
DNA ligation
catalyzed by DNA ligase
involves covalently bonding 2 broken DNA fragments / strands
DNA ligase
enzymes that catalyze forming bonds between 2 broken DNA fragments / strands
how to set up ligation reaction with Roche kits
add T4 DNa ligase, T4 DNA ligase buffer, water, vector DNA, and insert DNA to a tube
ligase goes last
let incubate at room temp for 15 min
T4 DNA ligase
catalyzes phosphodiester bonds between either blunt or sticky ends
T4 DNA ligase buffer
sets up binding conditions
has ATP so ligase can function
What is the best ratio of backbone and insert
1:1
what does gel extraction mean
taking DNA fragments from the band cut from the gel run and purify it so that the fragments can be used in the future
cut out band you want
put in microcentrifuge rube
add 1mL QG buffer
dissolve by putting in hot water bath
transfer to spin column and centrifuge
discard flow through
add QG buffer again and centrifuge and discard flow through
add PE buffer and centrifuge and discard flow through
spin empty
change outside of column to a clean microcentrifuge tube and add EB buffer
let sit before centrifuging again
function of QG buffer
solubility of agarose
used to dissolve the agarose from the gel
helps with binding to the column membrane
contains pH indicator (supposed to be yellow)
PE buffer
wash buffer to remove contaminants
EB buffer
elution buffer. anything bound to the membrane exits when spun down after using EB
what is the purpose of isopropanol
increases the yield of DNA fragments
add to PE and helps as part of the wash buffer
purpose of pH indicator gel extraction
tells you if the pH is too high
yellow = okay
orange / darker color = pH too high
absorption of DNA to silica depends on pH. 95% when pH is
pH < 7.5
efficiency of DNA is dependent of pH. The maximum elution is achieved at pH
between 7.0 and 8.5
open reading frame
sequence of DNA that starts with the start codon and ends with a stop codon
codes for a protein and is about 100 codon groups long
how to find open reading frame
find the start codon
what is the first amino acid residue of one orf
met (the start codon codes for it)
how does one orf stop after the last amino acid residue
when you reach stop codon
tell ribosomes that they’ve reached the end and to release the polypeptide chain created from the orf
function of blast
compare your query sequence to sequenced dna from their online database
important to compare because orf are conserved between proteins, so if your query sequence has an orf that’s the same as another sample, you can use it to try to figure out the purpose of the protein coded from the orf (and shape and all that stuff)
major steps of pfu-mutagenesis
mutant strand synthesis
DNP1 treatment to remove any parental plasmids
transformation using IPTG and X-gal
function of DPN1 enzyme
remove parental plasmid present after PCR
parental strands are methylated and new plasmids are unmethylated
function of beta-mercaptoethanol
precipitate DNA fragment
describe mutagenesis results
TAA - CAA
blue colonies, not white colonies
beta galactoside activity is restored
mutagenesis competend cells are different from regular transformation
ultra cold competent cells (even better competent cells)
you just gotta look at the separate cards for l6 japanese
sorry :)
