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1
Q

What is the purpose if first stop on a micropipette?

A

Allows you to uptake the desired volume accurately

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2
Q

What would you set the volume window to if you were pipetting 550 uL using a p-1000?

A

055

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3
Q

When uptaking the liquid into a micropipette, why must you slowly release the plunger?

A

Releasing the plunger too quickly will cause you to take up too much liquid which leads to inaccurate pipetting. It may also splash liquid into the pipette damaging it.

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4
Q

What would you set the volume window to if you were pipetting 200 uL using a p-200?

A

200

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5
Q

What would you set the volume window to if you were pipetting 90 uL using a p-200?

A

090

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6
Q

What is gel electrophoresis?

A

Gel electrophoresis is a technique used to separate DNA, RNA, or proteins based on their size and charge.

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7
Q

Why do you need a tip on a micropipette?

A

So the liquid does not go into the actual pipette and damage it

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8
Q

What is the purpose of the 2nd stop on a micropipette?

A

To expel the liquid from the micropipette

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9
Q

When loading a sample into a well of a gel, why must you keep the plunger depressed while pulling it out?

A

Keep the plunger depressed while removing the micropipette from the gel box so that you do not suck the liquid back up into the micropipette.

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10
Q

What would you set the volume window to if you were pipetting 1000 uL using a p-1000?

A

100

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11
Q

Centrifuge: Purpose

A

Used for the separation of fluids, gas, or liquid, based on density.

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12
Q

What volumes can a p-200 accurately pipette?

A

between 20 and 200 uL

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13
Q

What volumes can a p-20 accurately pipette?

A

between 2 and 20 uL

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14
Q

Describe proper balancing of tubes in a centrifuge.

A

Ensure tubes are balanced by using a “blank” (empty tube or tube with water) if needed.

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15
Q

What would you set the volume window to if you were pipetting 6.5 uL using a p-20?

A

065

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16
Q

What would you set the volume window to if you were pipetting 20 uL using a p-20?

A

200

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17
Q

Image of micropipette tip above gel wells

A

Illustration showing the correct position of the micropipette tip above the gel wells.

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18
Q

Centrifuge: How it works

A

Separation is achieved by spinning a vessel containing material at high speed.

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19
Q

How to load a sample into a gel using a micropipette

A

Position the micropipette tip above the well in the gel, but under the buffer solution.

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20
Q

In lab 4a, how many bands do you expect to see in the R+ lane? and what will be present in the bands?

A

2 bands, the smaller fragment is ~800 basepairs and contains pBAD and the RFP gene. The larger fragment is ~4500 basepairs, and contains the rest of the plasmid; this will include the araC gene, the ampR gene, and the origin of replication.

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21
Q

Purpose of loading dye in Lab 4a

A

Loading dye adds weight to the DNA which helps it sink into the well, it also allows you to track general movement in the gel box (it does not bind to the DNA so you cannot actual see the DNA moving, you can only infer that it is)

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22
Q

Which 2 Restriction enzymes did we use in lab 2a to digest the pARA-R plasmid?.

A

HINDIII and BamHI

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23
Q

What are “sticky ends”

A

Restriction enzymes like BamHI, HindIII, and EcoRI cut DNA at specific sequences, resulting in “sticky ends” which are single stranded overhangs that can be used for DNA recombination.

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24
Q

True or False: Restriction enzymes are naturally found in Prokaryotes.

A

True; Restriction enzymes provide protection against invading viruses by hydrolyzing “foreign” DNA.

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25
Use of restriction enzymes in LAB 2a
A combination of 2 different restriction enzymes is used to digest (hydrolyze) the pBAD-rfp segment out of the plasmid.
26
Overall Purpose of LAB 2a (restriction digest) and LAB 4a (gel electrophoresis)
Lab 2a and 4a are needed to ensure the plasmid is correct for making the red fluorescent protein. Lab 2a uses restriction enzymes to cut the plasmid, and lab 4a is used to verify that these are the correct size fragments for the para-R plasmid.
27
Purpose of DNA probe/stain in lab 4a
Probe binds to DNA, Allows you to see the DNA fragments under UV light which are otherwise clear
28
Purpose of LAB 4a
Use gel electrophoresis to separate out the DNA fragments; Examine the products from the restriction digest of the pARA-R plasmid
29
What probe did we use this year in lab 4a? Where was it located? How did it work?
We used GelGreen. It was mixed in with the agarose of the actual gel. When DNA is loaded into the wells and begins to migrate, it will pick up the probe from the gel as its moving. After, the DNA will glow green under a UV light of the trans-illuminator.
30
In lab 4a, how will you know there are Different forms of plasmids present including nicked, multimer, supercoiled, and "regular" plasmids.
The will move at different rates based on their size and/or shape. The multimer is the biggest since it is 2 plasmods connected together; it will move the slowest. The nicked, regular, and supercoiled are all the same size but will travel at different rates based on their shape. The supercoiled acts linear so it moves the fastest.
31
purpose of DNA Ladder
A DNA ladder is a molecular weight marker used to identify the approximate size of a molecule run on a gel during electrophoresis.
32
In lab 4a, what do you expect to see in the R- lane and why
You are expecting only full plasmids, but they may show up as separate bands or possibly a smear of DNA due to the different plasmid configurations (multimer, knocked circle, regular, and supercoiled)
33
How many Restriction fragments will there be after digest with Hind III and BamH I
2 fragments
34
In lab 2a, what was similar and different between the R+ and R- tubes
Similar: both the R+ and R- tubes had the pARA-R plasmids in them. Different: R+ tube had restriction enzymes in it and the R- tube did not
35
In lab 2a, what was happening in the R+ tube that was not happening in the R- tube?
Digestion using RE
36
Describe the parts of the pARA-R plasmid
The pARA-R plasmid is 5,302 bp long and includes components like the red fluorescent protein (rfp) gene, the ampicillin resistance gene (ampR), and the arabinose activator (araC).
37
Restriction enzymes cut DNA at specific recognition nucleotide sequences called ________
restriction sites
38
Step causing plasmid entry into bacteria (transformation step)
Heat shock causes the plasmid to enter the bacteria.
39
Lyse
To lyse means to break down the cell membrane, releasing the cell's contents.
40
Process of RFP expression involving arabinose and araC protein complex
Arabinose binds to the araC protein complex, preventing DNA looping (interaction with another site) and helping to align RNA polymerase on the promoter site (P-BAD). This process leads to transcription and translation of the RFP gene, resulting in RFP (red fluorescent protein) expression.
41
LAB 5a: Transforming Bacteria with the pARA-R Plasmid
Introduce the genetically modified plasmid into E. coli bacteria to produce the rfp protein.
42
Contents of the P- tube
The P- tube does not contain the plasmid (but it did contain E. coli).
43
Expected Growth of bacteria on plate containing Luria Broth, Ampicillin, & Arabinose (LB/amp/ara plate)
We only plated P+ on this plate. Only transformed cells should grow (the rest will die). The colonies should be pink since arabinose is the inducer to express the red fluorescent protein.
44
RFP
Red Fluorescent Protein, made from our gene of interest (the rfp gene)
45
LAB 6b: Separate the red fluorescent protein with column chromatography
The hydrophobicity of RFP was used to separate it from other proteins using column chromatography and buffer solutions that decrease in salt concentration.
46
centrifugation process in lab 6b
Since the cells were already lysed open in lab 6a prior to this, once centrifuged, the cell pellet containing heavy cell membranes and cell debris sink to bottom (the pellet in our lab was white), and the less dense liquid remains on top (this is the supernatant which contains the released red fluorescent proteins).
47
Identifying transformed bacteria on petri plates
Transformed bacteria can be identified by their growth on a plate that contains antibiotics. (untransformed cells would die)
48
Three buffers are used to separate a highly hydrophobic protein in a column.
Binding buffer, wash buffer, and elution buffer are used in column chromatography to separate proteins (in that order).
49
How did we get the different proteins to stick to the resin of the column in lab 6b?
For hydrophobic amino acids to stick to the resin, proteins must be unfolded to expose these amino acids, which are usually inside the protein. We used buffers of various salt concentrations to scaffold these shape changes.
50
LAB 6a: overnight culture
Transformed E. coli cells are grown over night in a shaking incubator which agitates the cells and maintains the ideal temp for cellular processes such a DNA replication, protein synthesis, and mitosis. The were grown in a solution contain luria broth (food), ampicillin (antibiotic to kill untransformed cells), and arabinose (to allow expression of rfp).
51
Expected Growth of bacteria on plate containing Luria Broth & Ampicillin (LB/amp plate)
The P+ side of the plate should contain white colonies. These are only the transformed cells (they have the plasmid which contains the ampicillin resistance gene so they live in the presence of antibiotics). The P- side of the plate should not have growth. The bacteria did not get any plasmid so they are not anti-biotic resistant, so they will all die on this plate)
52
Description of Recombinant plasmid of interest: pARA-R
The pARA-R construct is a recombinant plasmid with a size of 5,302 base pairs. It includes sections such as araC, AmpR, PBAD, and rfp, with restriction sites BamHI and HindIII.
53
Wash Buffer (Lab 6b)
Wash Buffer (WB) had a medium salt concentration and was the 2nd buffer used. It washes out the proteins that are slightly less hydrophobic than RFP, and keeps RFP stuck to the resin in the column.
54
Contents of the P+ tube
The P+ tube contains the plasmid (and E. coli).
55
cell lysis and centrifugation process in lab 6a
Lysis Buffer was used to disrupt the cell wall and membrane to extract cell contents, followed by centrifugation to separate components. Dense cells (making rfp) sink to bottom, lighter liquids (containing LB/amp/ara) stay on top.
56
Expected Growth of bacteria on plate containing only Luria Broth (LB plate)
Both the P+ and P- sides on the plate should shown bacterial lawns (unrestricted growth; LOTS!)
57
Environmental needs for transformed bacteria to make rfp protein
Transformed bacteria need Arabinose (an inducer) in their environment to make rfp protein.
58
Binding Buffer (Lab 6b)
Binding Buffer (BB) had the highest salt concentration and was the first buffer used. It rotates the hydrophobic R-groups of the amino acids to point outside the RFP cylinder (hydrophilic R-groups are rotated inward). This allows for hydrophobic proteins to remain stuck to the hydrophobic resin (such as RFP), while washing out the hydrophilic proteins from the column.
59
Column comes with equilibration buffer (CEB).
The equilibration buffer prepares the column for the separation process. FYI: This is the least important of all the buffers we used and will not be on the test.
60
Elution Buffer (Lab 6b)
Elution Buffer (EB) had the lowest salt concentration and was the 3rd & final buffer used. It rotates the hydrophobic R-groups of the amino acids to point inside the RFP cylinder (hydrophilic R-groups are rotated outward). This allows for the RFP to be released from the column. This is the purified protein that we collected! FYI: to elute means to release, so the Elution Buffer elutes the rfp from the column.
61
Type of resin used in column chromatography for lab 6b
The column is packed with a hydrophobic resin that attracts hydrophobic amino acids.
62
What makes our E. coli competent?
The have been coated in Calcium ions (which leads to adhesion zones allowing for the plasmids to potentially enter)
63
buffers in lab 6b (general)
Buffers are salt solutions that unfold proteins to different extents (BB, WB, EB).
64
What type of bond is found between the monomers of arabinose?
Glycosidic
65
Which site (loci) on the pARA-R plasmid is the promoter for the rfp gene?
pBAD
66
What was the purpose of arabinose on the plates in lab 5a?
Sugar to allow the expression of the rfp gene
67
Which buffer has the lowest salt concentration?
elution buffer
68
What is the gene of interest on the pARA-R plasmid?
rfp
69
Which buffer has the highest salt concentration?
binding buffer
70
What group of organic macromolecules do restriction enzymes belong to? what are the monomers called?
proteins, amino acids
71
What gene on the pARA-R plasmid is the antibiotic resistance gene?
ampR
72
What group of organic macromolecules does arabinose belong to? what are the monomers called?
carbohydrates, monosaccharides
73
what is the formula for transformation efficiency? units?
of counted bacterial colonies / amount of DNA spread on the plate, cfu/ug
74
what step causes transformation?
heat shock
75
What gene on the pARA-R plasmid makes a protein that acts as an inducer for the red fluorescent protein?
araC
76
Which site (loci) on the pARA-R plasmid is the place of origin?
ori
77
What type of bond is found between the monomers of restriction enzymes?
peptide
78
what makes the E. coli cells used in lab 5a competent?
they are coated in calcium ions

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