Biology Lab Exam Review

What does the lysis buffer contain?

SDS detergent and EDTA

What is the purpose of EDTA?

removes Mg2+ ions, destabilizing the cell wall and the cell membrane

What does SDS detergent do?

breaks up the cell membrane by removing lipid molecules from the membranes

Why did we need to cut the plant leaves with a scalpel?

to begin the physical breakdown of the plant to ensure we extract the highest concentration of DNA

What was the purpose in adding ethanol to the supernatant before you loaded it into the column?

the ethanol helps the DNA bind to the column

What is the difference between the results of DNA extraction and those at the end of PCR?

DNA extraction: the entire Jacaranda gDNA

PCR: only the GAPC genes

Once you bind the DNA to the silica membrane filter, during centrifugation what macromolecules flow through the column?

Lysis buffer, plant debris, proteins, polysaccharides, and lipids

What is the purpose of doing 3 wash steps during the column purification?

To ensure that all the contaminants (polysaccharides and proteins) were washed away

Why do you incubate the column with water heated to 70 degrees for one minute before you elute it off the column?

It helps dissolve the salts and DNA, breaking the bonds easier.

Why does DNA elute off the column with water and not with a salt solution?

the salts bind the DNA to the silica membrane; water breaks the bridge and carries DNA through the column

Where is the DNA after elution?

in the new collection tube

Why was it necessary to change collection tubes and have one that has a cap for elution?

Because the DNA is eluted in the last step, we can ensure that the DNA is free of any outside contaminants with a new collection tube.

What are the different controls used in degenerate PCR and their purposes?

water: negative control that tests for contamination

pGAP: positive control that tests if the PCR reagents and thermocycler are working

Arabidopsis gDNA: positive control that tests if the primers are working

What is the purpose of setting up two positive controls? What is the difference between these two?

pGAP will replicate in our PCR without primers because it has its own origin of replication. However, Arabidopsis gDNA requires primers in order to undergo PCR.

What is the purpose of setting up a negative control? What would it mean if you saw a band?

A band in the negative control would indicate that our sample was potentially contaminated and that our results cannot be easily trusted.

Why did you need to change your tips each time you pipetted any solution during the PCR setup?

to prevent any cross-contamination between samples.

What were the components in the complete mastermix?

Primers, dNTPs, Taq polymerase, pH buffer, salts

What specific steps did you need to take to calculate how much primers and master mix to pipet together?

C1V1 = C2V2, as well as knowing that we need to make the amount needed +1.

Why did you need to mix the PCR reactions by flicking the tube and then do a 5 second centrifugation before you placed the reactions in the thermocycler?

To ensure that they are all at the bottom of the tube and homogeneously mixed.

What were the specific thermocycler conditions for degenerate PCR, and why?

95 - denaturing

52 - annealing

72 - extension

Briefly explain the process of PCR. How does it eventually amplify the region of interest?

By using forward and reverse primers made from the GAPC region of the Arabidopsis plant, we only amplify the regions in between these two primer regions and hypothetically therefore only amplify GAPC genes.

Why do degenerate PCR primers sometimes amplify regions that you don't want?

Because the degenerate primers contain wobble regions that are unspecific, and also because we are using the entire gDNA sequence that may have regions that also match our primer sequence.

What is a degenerate primer?

A primer that has unspecific wobble regions

If you needed 20uL complete mastermix per PCR reaction and wanted to run 4 reactions, how much mastermix would you prepare?

100uL

What charges are at each end of the gel and why?

top is negative

bottom is positive

BECAUSE - DNA is negatively charged and is attracted to the positive end, needs to move down the gel.

What would happen if you switched the charges on the gel?

the DNA would move in the opposite direction and leak outside the top of the gel.

What is the difference between loading dye and ethidium bromide? What is the purpose of each?

loading dye: similar in size to DNA, shows during electrophoresis any movement and if the sample was loaded into the well correctly. Also weighs down the DNA

ethidium bromide: see DNA fragments; binds to DNA so we can see the results under UV

What compound in gel electrophoresis binds to DNA?

ethidium bromide

What is a molecular ladder?

known lengths for reference for PCR

What is the gel you used made of?

agarose

Explain why you cannot use water to run the gel

Water can't be used to run this gel because the buffer is a better conductor of electricity. Additionally, the agarose gel we used would dissolve in water.

Why does DNA get attracted to the positive pole when placed in an electrical field?

because DNA is negative

Why did you not treat the degenerate PCR reactions with exonuclease before you ran the degenerate PCR?

We needed the degenerate primers in order to successfully run PCR (exonuclease would have digested all the primers and PCR would not have worked).

What is the goal of running exonuclease digest? What would happen if you forgot this step?

The goal is to remove any degenerate primers in our solutions. If we forgot this step, then the degenerate primers would still bind to the DNA and impact our nested PCR results.

Why wasn't the initial pGAP positive control treated with exonuclease?

We used a new pGAP positive control - it would have been unnecessary

What would happen if the exonuclease was not heat inactivated?

It would also digest our nested primers and therefore we would have no change in our gels after nested PCR.

Why was the initial negative control reaction carried through to nested PCR?

to test to see if there was any contamination during degenerate PCR or any steps leading up to nested PCR.

Why wasn't the initial positive control pGAP carried through to nested PCR?

pGAP is used to test if the thermocycler is working; if the original pGAP is already replicated via the initial degenerate PCR, we cannot accurately tell if the thermocycler works during nested PCR.

Why were both of your initial plant PCR reactions carried through to nested PCR?

We started off with degenerate PCR results from Jacaranda, which have the possibility of being GAPC1/2, GAPCP1/2, GAPA, GAPB, and GAPN. Because the nested primers are within the GAPC genes, it will help narrow down the types to only GAPC1/2

Why was a new negative control reaction made for nested PCR?

To test for contamination for nested only

What are the components in each of the two negative controls?

old: old negative control + MMIP

new: DI water + MMIP

How was the mastermix for this round of PCR different from the mastermix for the initial PCR?

nested primers vs degenerate primers

When could contamination have occurred had you seen a band in your negative control that used the carried over negative as template, but not in the new negative with water as template? Explain.

degenerate PCR or steps leading up to nested PCR (such as exonuclease digest)

Why did you need to dilute most of your samples?

to reduce the concentration of non-specific sequences to increase the chances of replicating GAPC

What steps did you take to dilute your samples? What was the final ratio

1: 98uL water + 2uL sample

2: 20uL dilution 1 + 20uL MMIP

total = 1/100 ratio

Why wasn't it necessary to dilute the new negative control or new plasmid control?

negative: nothing to dilute because there are no products (only water and MMIP)

pGAP: we are using a new, pre-diluted pGAP

What were the thermocycler conditions for the nested PCR reactions,?

95 - denaturing

46 - annealing

72 - extension

Why does the nested PCR reaction have a lower annealing temperature compared to the degenerate PCR?

to promote more primer binding now that our primers are more specific to the sequences.

Why could we not just pipet 0.4uL of the initial PCR sample in a final volume of 40uL to make the dilution?

the amount is too small, leading to inaccurate results and increasing the chances of obtaining a non-specific sequence.

What were the expected size ranges of the target GAPC/GAPC2 PCR products?

1-1.5kb

What are the small bands below the last fragment of the ladder?

primer-dimers: self-annealing of the primers used during PCR

Why was it so important to use a centrifugation of 0.7g with the small centrifuge instead of full speed for the spin kleen columns?

High centrifugation speeds would crush the very delicate polymer beads and also would force everything through, making our size-exclusion chromatography useless.

Why was the spin kleen column centrifuged for 2 minutes at 0.7 g before the PCR reaction was added?

to ensure that all of the polymer beads were tightly packed and uniform with each other

Why were the first round PCR reactions not purified using the PCR spin kleen columns?

They contain undesirable sequences.

Briefly explain how size chromatography works and how the purified PCR product was obtained. Why did it not stick to the column?

-porous polymer beads were added to the column

-small molecules, dNTPs, and small sequences get stuck in the beads

-larger molecules including gDNA and GAPC do not fit inside the pores and move around the beads, eluting through the column due to the centrifugation

State what sticks to the PCR spin kleen columns after the PCR reaction is added and spun for 2 minutes.

Primers, primer-dimers, dNTPs, Taq polymerase

Why was it important to change collection tubes when PCR sample was added to the column?

Because the desired product elutes into the column, we want to ensure that there is no contamination from the previous spins

What would happen to the cloning experiment if the proofreading reaction was done after the ligation reaction?

the ends would still have the hanging A and would not ligate correctly to the plasmid.

What bond in DNA does the ligase enzyme catalyze?

phosphodiester bonds

Name one advantage of sticky end ligation over blunt end ligation.

-more accurate binding

-sticky ends have directionality

Why was it important to mix the contents in the tube and then do a quick centrifuge when the proofreading reaction was setup?

it mixes the contents and forces it to the bottom

Why did you need to do a proofreading step?

We needed blunt ends to match our pJET plasmid

What would have happened if you had skipped the proofreading step?

The insert would not ligate to the plasmid vector

If a different plasmid was chosen to do the sticky end cloning and the 3' ends of the insert were left with sticky end As, what would be the vector sequence that would anneal to those As?

vectors that have a sticky end Ts

Why did you not work with any living organisms this week during ligation?

that week's only purpose was to proofread the GAPC fragment and ligate it to the plasmid vector -> we are not trasnforming bacteria yet

State the reason why plasmids have an origin of replication.

independent reproduction (self-reproduction)

What does it mean that plasmids have a MCS?

Multiple cloning sites are within a plasmid that allow for simpler insertion of DNA since the insert can be added within any site. They have unique restriction sites where restriction enzymes will cut and an insert can be added.

Why was the intensity of the nested PCR product checked on the gel before starting the proofreading reaction? To what DNA sample and specific band was it compared? Be specific.

to check if there was enough DNA to insert into the plasmid to ensure a high yield of successful transformations

Explain why it is important to have a molar ratio for ligation that is 1:1 or more vector to insert

too much vector - self-ligation

too much GAPC - double insert/primer dimers

Why is it said that doing blunt end ligation results in no directionality?

The plasmids can be inserted into the plasmid forwards, backwards, or upside-down since there are no sticky nucleotides that it needs to match.

What is the importance of growing bacteria on plates containing Amp and IPTG?

Amp - removes untransformed E. coli by killing it

IPTG - acts like lactose to induce expression of plasmid genes via the lac operon

What phase of bacterial growth must cells be in to become competent?

exponential growth phase

What does it mean for bacteria to become competent?

It must be able to uptake foreign DNA

Why are cells grown for 20-40 minutes from an overnight culture?

It ensures that we catch the bacteria in the growth phase to yield high competency

What would happen to the transformation efficiency if the cells were left at room temperature while making them competent?

They would be far past the exponential growth phase, lowering competency

Why were the plates incubated overnight at 37C after transformation?

It allows surviving colonies time to grow

When the cells were made competent, why was it important to resuspend the pellet and not have any clumps before proceeding to the next step?

so bacteria had equal exposure to the plasmids and buffer

Why was it very important to only take out one plate at a time when the heat shock was performed?

It is important to keep the plate as close to 37C as possible to ensure maximum transformation potential

What would happen if the LB+Amp+IPTG plates were kept growing in the incubator for an entire week and never put in the refrigerator?

colonies would keep growing and we would not be able to tell the difference between colonies

your friend states that after transformation this week you inserted the plasmid into the nucleus of E. coli. Why is this incorrect?

bacteria do not have nuclei

What would be the possible explanations if no colonies grew on the plate?

- we did not properly heat shock the bacteria meaning

- no inserts were successfully able to transform into the vector

What are satellite colonies?

small bacteria colonies w/o plasmids that grow due to low AMP surrounding larger colonies

Why were the cells resuspended in 300 ul TF buffer, but then following the second centrifugation, the pellet was only suspended in 150 ul?

300uL - lots of exposure to TF buffer

150uL - higher concentration to allow for more contact

What would happen if the cells were kept out of ice for 5 min while waiting to use the centrifuge as the competent cells were prepared?

The cell likely would move past the exponential growth phase and therefore would likely not become competent

What are the components of transformation buffer and why?

CaCl2 - DNA binds to the cell wall

DMSO - disrupt the cell membrane

What would happen if the diluted cells grew for 20hrs instead of 20 min?

cells are now long past the exponential growth phase

at what temp is exonuclease I inactivated

80 deg C

at what temp is exouclease I activated

37 deg C

what temp does degen pcr anneal at?

52 C

what temp does nested pcr anneal at?

46 C

why does nested pcr anneal at a lower temperature?

to encourage binding