Show:
cDNA Library
PCR and Restriction Digest
Miniprep
DSAP
Misc.
100
Why do we use cDNA instead of mRNA or plant DNA?
cDNA is more stable and it only includes the exons, or the coding region of the DNA.
100

What is another name for restriction enzymes?

Endonuclease

100
Miniprep is done after which lab?
PCR and Gel Electrophoresis of the PCR
100
What does DSAP stand for?
DNA Sequence Analysis Program
100

What direction is DNA synthesized in?

5' --> 3'

200
What Bacteria is used in our cDNA library?
e. coli
200
What number do you subtract from the RD gel
700
200
What is the proper way to resuspend a pellet?
Using a clean pipette tip, gently pipette up and down until the pellet breaks apart and is evenly distributed throughout the solution.
200

What is the sequence of DNA we search for when trying to find the beginning of the sequence?

GGCCGG

200
If I have to pipette 100 uL of solution, which pipettemen should I use?
p200
300

What is a partial digest and why do we need it?

cuts fragments into uneven, overlapping sections so we can put together the genome of an organism by finding overlaps in the sequences

300
What is the function of primers?
DNA polymerase must have something to attach nucleotides onto, it cannot synthesize DNA chains by itself.
300
What is the purpose of the wash?
It gets rid of cellular debris in the spin column.
300
What is the difference between blastn, blastx, and blastp?
Blastn searches the databases for matches to the query nuclieotide sequence. Blastx searches for amino acid sequences for matches to the query nucleotide sequence BlastP searches for amino acid sequences for matches tot he query amino acid sequence
300

What is the infamous carcinogen dye that we work with?

EtBr

400
When extracting the mRNA from the plant, how do we tell the mRNA from the tRNA and rRNA?
We use strings of T's in order to catch the polyA tails of the mRNA
400
A PCR gel has 4 bands, how do we determine the size of the insert?
It cannot be determined. It is contaminated.
400
What is in the pellet and what is in the supernatant after the first spin?
Bacteria is in the pellet, LB is in the supernatant.
400

What is a nucleotide-to-nucleotide search and why is it not fully reliable to find matches?

nucleotide-to-nucleotide compares the DNA sequence data with all other DNA sequences data that is stored in databases. unreliable because DNA sequences that code for same protein can have completely different DNA Sequences due to degeneracy of genetic code

400
What are two functions of the loading dye?
1. track how far the DNA ran 2. weigh down the DNA so it does not float out of the well
500
Explain why there are white colonies and why there are blue colonies.
Our bacteria has a gene called LacZ which codes for an enzyme that breaks down sugar. When the sugar is cleaved, a blue color appears. Since our sequence is inserted inside of this gene, it interupts the production of this enzyme. Since there is no sugar cleaved, the colony remains white. Which means white colonies are the colonies that has our sequences.
500
A RD gel turns out to have 4 bands. How do we determine the size of the insert?
the band closest to the top of the gel is the vector, so that can be ignored since it is not part of our insert. determine the size of the remaining 2 bands and then add the sizes together since they are part of our insert but were separated because of internal restriction sites.
500

What is P1 buffer made up of, and what purpose do each of the components serve?

- EDTA - breaks outer membrane of the cell and intracellular enzymes (gets rid of DNA cleaving enzymes)
- Glucose and Tris - buffer the pH
- RNAse - removes and degrades RNA fragments (these make it hard to purify DNA)

500
What are three things that indicate your sequence code for the start of the protein?
-sequence starts with M -Blastx matches with the 1st M of the database sequence. -Blastp matches with the 1st M of the database sequence.
500
If a gel turns up totally blank, what can be wrong with it? What can we do to fix it?
1. The electrodes were connected the wrong way, causing the DNA to run off the well. Do a new run. 2. Someone forgot to add EtBr to the gel. Soak the gel in EtBr and then look at it again.