BIT 473 Lab midterm Notes

Please study Exam Feb 18

What type of Reaction does the Alkaline phosphate do ?

Known to remove the Phosphate Group to different molecules Through catlazyng the hydrolisis of PNPP(P-nitrophenyl Phosphate).

What was the independent variable when you were determining the K_m and V_Max of an enzyme ?

Known as the Substrate concentration PNPP (P-nitrophenyl Phosphate).

What is the difference between the D1s80 alleles ?

Known as the Number of Tandem repeats

In PCR, what happens during elongation?

Known as the DNA polymerase copies the Target sequence

In PCR, what allows to copy a specific sequence of DNA ?

The sequence of the primers

What is the most ideal conditions for E.coli DNA polymerase ?

Known to be at 37 C

What are the Key steps of the PCR process when analyzing the DNA ?

Known to be Denaturation At (94 c), and annealing at 65 C and lastly Extension at 72 C

Importance of nucleotide sequence.?

Must be placed at either end of the target DNA for the Known Primers to be accurately generated.

Description / issues with PCR?

PCR or Polymerase chain reaction Is very powerful and Sensitive technique through the use of contaminating DNA as well of changes in the pH and concentration of the necessary components can alter the results in a False positive outcome

Where does the D1s80 Locus appear and describe it

Known to appear as the Short arm within the Chromosome #1 And is known to be comprised of a series of tandem repeats of a 16 bp (base pairs sequence).

Uniquess of the TAQ DNA polymerase ?

Know to provided stability when place into a high temperature due to its bacteria content can cause the enzyme to have optimal activity at 72 C. At a higher temperature increase at 95 C can be know to remain its activity for about 25-30 cycles.

Issues with too small and to large primers

If primer too small will result in the template to bind back on itself causing the self primer. To much primers may lead to binding within itself (creating primer dimers) and to other locations within the template.

What us a VNTR?

Deemed as a Locus with a Variable Number of the same sequence.

Why do you need to boil the Agarose while preparing an agarose Gel ?

Helps with dissolving the agarose.

What is the molar extinction co- efficient for Nitrophenol formation?

Is deemed to be E₄₁₀ =18 400M^-1 Cm^-1

Describe Plasmid ?

they are deemed as Extrachromosomal and small circular DNA molecules that could be isolated as intact molecules

Define The use of EDTA in Alkaline Lysis ?

Its know to bind with the divalent cation that are necessary for cell membrane Stability.

Describe SDS and its use

Known as sodium Dodecyl sulfate (SDS) acts as an ionic detergent know to dissolve the phospholipids and protein components of the cell membrane.

Use of the NAOH in Alkaline lysis Process

Know to be used to denature both plasmid and chromosomal DNA into single strands.

Use of 95% ethanol in Alkaline lysis ?

Can carry a cation of K⁺(potassium) charged will cause the cation to bind with the negative phosphate backbone of the DNA. This causes the DNA Molecule to repel one another and therefore to precipitate

Describe the Spin columns within the process of Plasmids DNA isolation?

The spin column contain a Silica-based resin which binds nucleic acids in the presence of a chaotropic salt. The chaotropic salt disrupts non-covalent interactions between molecules and weakens the affinity of nucleic acids for water.

Describe an Issue that causes multiple Bands occurring from a DNA sample onto PCR Agarose Gel ?

Keeps binding to every single Repeat. That will be seem numerous to create multiple bands to appear in the PCR gel. Designed to be complimentary to the repeats

Notable Trait within the Plasmid DNA ??

Should provide a single recognition site for one or more restriction enzyme in the region of the plasmid not required for the replication(of cloning the DNA)

What can be used to make the artificial Bacteria Transformational ?

Can be known through as the process of using the divalent cation Mg²⁺ or Ca²⁺Or using the approach

of electrical shock called the Electroporation.

Effects of process using the Divalent Cations ?

They can help to stabilize a molecule such as E.coli Plasma as well as the Other membranes allowing its cell envelope to become more permeable towards the DNA molecule. As well the Ions more specifically the Ca²⁺ Shied the negative charges on the bacterial cell surface which prevents the repulsion of the (-) Negatively charged DNA molecule.

Describe the Purpose/ Use of the Chaotropic salts ??

Known to be found in solution (III) Can cause disruptions to appear between non covalent interactions Between the molecule and weakens the affinity of nucleic acids With Water. When washing Columns with ethanol the salts get removed with other contaminates

Evaluating a DNA purity ?

Known as the absorbance begins being measure from the wavelength of 230 nm to 320 nm. A good quality DNA with have a ratio Of A₂₆₀ / A280 ratio of about 1.7 -2.0. Having the data lower than 1.6 Indicates more contamination that is present. A reading greater than 1.8 suggest contamination by RNA.

Where is RNA absorbance read at and what can affect it ??

Know to be at 260nm as well the molecule of guanidine will make higher 260 nm absorbance can affect calculations for yield making DNA quality over estimated ,

Briefly explain restriction enzymes ??

Are known to be apart of the Bacteria Restriction modification systems. They can be described to prevent and restrict Invasion of Foreign DNA. Such as Viral DNA, By cutting it up .

How are primers Created ?

Are founded at either ends of a DNA within a nucleotide sequence

Briefly describe the D1s80 locus??

Are known to appear as short arm On chromosome #1 for humans, Its know to be composed of Tandem repeats with a 16 bp sequence.

Identify and Explain the (VNTRS)?

will be known as the variable number of tandem repeats which can be the arrangement of the tandem repeats. The different alleles of this locus will have differing number of this 16 bp sequence.

Correlation Of D1s80 within the PCR ?

Know to work in part with primers that designed to the unique DNA sequence that will flank the D1s80 locus. The PCR allows the amplification of the DNA between and including the primers different alllels correlate to PCR product difference in size

Briefly explain transformation of the fragile cells?

After undergoing several process it will recover its stability and begin to express any genes carried by the Transformed DNA , Including Antibiotic resistance Gene

Describe the Transformation efficacy?

Will be a fraction of the cell that they can take up The DNA. It is expressed as the # of transformed cells per μg of the DNA

Analyzing result with an Agarose Gel in regards to enzyme digestion

No digestion will show a big clump /large Band appearing on the Gel.

Partial Digestion will have to bands appear but some distance away and have a Lighter less Defines band appear.

Full digestion appears as One solid Define Band appearing with no contaminants or any debris nearby it.

Which solution will be causing the complete lysis of bacteria in the alkaline lysis method.

Solution #2 - Through the use of the SDS An ionic detergent will end up dissolving to phospholipids and protein components that lie within the cell membrane. And to be lysed and released to the cell

Which of these absorbances indicate contamination from Chaotropic salts ?

Known to be at 230 nm

Which step in Alkaline Lysis is replaced by the use of a column in the DNA extraction Kit ?

Known as the Precipitation of plasmid DNA with ethanol

Which of the following moleucles help with the biding of DNA to the silica in the columns ?

The Chaotropic salt that allows the disruption of non covalent Interactions between the Molecules and to weaken the affinity of nucleic acids For water.

Describe the early Log phase In bacterial transformation?

Deemed to be the higher amount of transformation efficiency helps with each step recover as well as allow the Antibiotic Resistance gene expression to be activated.

In chemical transformation, what helps neutralizing the charge of DNA and the cell membrane?

Known as Ca²⁺ Calcium

Which of the following treatments will you use to make the bacterial competent ?

With Ca²⁺ and Mg²⁺

Which Antibiotic does Puc18 Provide resistance to ??

Know as Ampicillin .

How does the Visualization Of DNA appear on an agarose Gel ?

Known as Using a DNA binding florescent Molecule

Which of the following Reagents helps with the Washes of the column when isolating the DNA ?

Deemed as Ethanol

How does Red safe Make the DNA visible ??

It binds to DNA and Absorbs UV/Blue light on its own.

Which of the following is different in plasmid DNA isolation by alkaline Lysis comapred to the Isolation with a Kit ?

Through the use of Chaotropic slats

In the Lac operon experiment Which of the following wave lengths did you use to follow the bacterial Growth ?

Known as 550 nm

What does IPTG simulate in your experiment Of lac Operon?

Known as the Hugh sucrose molecule

How does the cAMP activate the Lac Operon ??

Known as activating the CAP protein

What is the Goal of the β-Galactosidase within the Lac operon experiment

Know to be The tool to study The allosteric activation of the enzymes

Which of the following Inhibition Mechanism would alter the K_m of an enzyme?

Known as The completive Inhibitor

Result/ Viewing changes within a Non-completive Inhibitor of an enzyme ?

Deemed to have A descrease in Vmax Meang the speed of the Reactions slows down And contains some binding that is inactive.

The K_M will be unchanged

In an SDS-Page, what do you need to add to polymerase the acrylamide?

Both APS as well as TEMED

meaning/ Use of Idienufitying the Reaction rate ?

Can be a Useful method that can determine the amount of substrate or product that is called an ASSAY ?

What Is the simplest way to Activate a Gene within the Lac Operon ?

Known as being Tasked with increasing or upregulate its transcription into mRNA.