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
When in doubt, who do we email?
SUE.
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?
CGGCCGGG
200
If I have to pipette 100 uL of solution, which pipettemen should I use?
p200
300
What is the sterile technique called when we are picking our colonies?
"Clamshelling"
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 that waksmanite must 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
On CN3D, which "rendering shortcuts" best show the alpha helices and beta strands?
Worms
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
Explain the roles of solution I, II, and III.
Solution I resuspends the bacterial cells. Solution II breaks open the cells as well as the DNA and plasmids. Solution III allows the DNA and plasmids to come together again; however, only the plasmids will return to its normal shape since it is much smaller and closer together than the 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.
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