Quiz 1 Info Contd 3

Question 1

spectrophotometer reading gives number of … we isolated and relative … compared to everything else that was originally in the isolation - want to see increase of … relative to other components

Answer 1

nucleic acids;
purity;
DNA

Question 2

DNA absorbing at … nm
… ratio comparing amount of nucleic acid to protein
… ratio is comparing DNA to other things that were in buffers, for instance

Answer 2

260;
260/280;
260/230;

Question 3

For the 260/280 and 260/230 ratios, between … and … is favorable

Answer 3

1. 8;

2. 2

Question 4

… buffer in gel: helps with having the … that’s there

… in gel as well - … and shows where we have DNA in the gel

Answer 4

TAE;
electric field;
ethidium bromide;
fluoresces

Question 5

two main bands in gel for plasmid - nicked band and supercoiled:

• … should ideally be more prominent, migrates further
• for the other one, one of the two strands has a …, allowing DNA to …

Answer 5

supercoiled;
break;
untwist

Question 6

two main bands in gel for plasmid - nicked band and supercoiled:
- supercoiled is twisted back on itself and is more … and …, moves through gel as if it’s … than it actually is

Answer 6

dense;
compact;
smaller

Question 7

linear DNA would seem smaller than … but not as small as …

Answer 7

nicked;

supercoiled

Question 8

if there’s anything that moves faster than the supercoiled band - it’s …, …

Answer 8

circular, single stranded DNA

Question 9

… polymerase - can only use DNA

Answer 9

DNA dependent DNA

Question 10

Prep:
make reverse transcription reaction and make RNA sample (RNA + primer + water)
also have to heat up the RNA to 70 degrees C to … and get rid of any …

Answer 10

denature the RNA;

secondary structure

Question 11

mix reverse transcriptase and RNA sample and let them … and then switch temp from 25 degrees to 42 degrees C

• 42 is where … becomes active, and … step occurs
• at 70 degrees, … will be inactivated

Answer 11

anneal;
reverse transcriptase;
extension;
reverse transcriptase

Question 12

gene specific primer only binds to that particular gene, only works well if sequence is … Otherwise, may not be able to bind anything.
… primer will bind to anything that has a … on it
… primer: 6 nucleotide primer with random sequence

Answer 12

well expressed;
oligo dT;
poly A tail;
random

Question 13

+RT is the experimental tube: contains … and …

Answer 13

RNA template;

reverse transcriptase

Question 14

For -RT, volume of reverse trans is replaced with …

• this is done to make sure that we’re not copying from … or any other … in the sample
• shouldn’t see product in … If there is a product, tells us that there’s … contaminating the sample

Answer 14

water; 
genomic DNA; 
DNA; 
PCR; 
genomic DNA

Question 15

Shouldn’t see PCR product for -RNA either. If there is a product, indicates that one of the other components is …
~
… controls bc we’re not expecting to see products in PCR for them

Answer 15

contaminated;

negative

Question 16

28S and 18S bands and small RNA bands for … and … on the gel

• see RNA in … and …
• No RNA should be present for …

Answer 16

+RT; -RT;
+RT; -RT;
-RNA

Question 17

smear for … should be brighter than that of …

should be no enhancement of nucleotide signal in ..

Answer 17

+RT; -RT;

-RT

Question 18

these products that we get are … for PCR

… for PCR - product size will be about … bp; should see nothing for -RT and -RNA on gel after PCR

Answer 18

templates;
linear ds RNA;
1141;

Question 19

linear ds RNA for PCR
if genomic DNA in -RT, would see band at about … bp
if -RNA was contaminated, could see bands … along the gel

Answer 19

2500 bp;

anywhere;

Question 20

we’re trying to amplify the …, makes it important to know where start and stop codons are
if trying to amplify specific domain, this wouldn’t be that important

Answer 20

whole coding region

Question 21

know which primer is … and which is …
primers are …-… nucleotides in length bc they’re very specific –> highly unlikely to just randomly find that sequence

Answer 21

upstream;
downstream;
18; 24;

Question 22

want to have primers with …-…% GC content
keep GC and TA content relatively …
higher annealing temp - …, can’t …
lower annealing temp - primers start … (I think - check)

Answer 22

40; 60;
equal;
less primer binding; copy anything;
binding anywhere

Question 23

… helps 3’ end bind to template and polymerase starts copying from end
having more than … Gs or Cs can be problematic

Answer 23

GC clamp;

2 - 3;

Question 24

if primer forms …, can’t form template

if primer … with itself or with other primer, won’t be able to form template

Answer 24

hairpin;

hybridizes

Question 25

avoid … and … - having same nucleotide … or have same set of …

Answer 25

runs;
repeats;
repeated;
two nucleotides

Question 26

avoid runs and repeats - having same nucleotide repeated or have same set of two nucleotides
- by having repeated units, primer can … on template and won’t start from exact same place

Answer 26

slide