Quest 4 Flashcards
_________ is the final method to absolutely confirm the identity of the insert in your plasmid DNA and screen for potential mutations.
DNA sequencing
If the sample is pure enough, the ______ applies
Beer-Lambert Law
(A= EdC
Absorbance = wavelength coefficient x distance x concentration)
DNA and RNA absorb UV light most strongly at ____.
260 nm
Protein absorbs UV light most strongly at _____.
280 nm
____ is the most common contaminant.
Protein
Pure preparations of DNA have OD260/OD280 values of _____.
1.8-2.0
If there is contamination with protein, this ratio will be ________ and accurate determination of the amount of nucleic acid is not possible.
significantly less
We used a ________ to measure DNA concentration.
Nanodrop Lite
What are the steps to blanking the machine?
- Lift the arm and pipette 2 ul EB onto the lower pedestal.
- Lower arm and press “blank”
- Lift arm and wipe the upper and lower pedestal with Kimwipe
- Repeat to confirm the blank
What is the purpose of blanking the machine?
It cancels out any absorbance by any other substance other than the molecule of interest.
We eluted with elution buffer so we have to ____ with elution buffer.
blank
(You blank with whatever substance is your background substance)
What are the steps for loading the miniprep samples onto the machine?
- Lift the arm and pipette 2 ul mini-prep onto the lower pedestal
- Lower the arm and press “measure”
- Lift the arm and wipe the upper and lower pedestal with Kimwipe
- Record the A260, A260/A280 ratio, and DNA concentration
To calculate DNA concentration, use this formula:
OD260 x 50 ng/ul x dilution factor
(In ug/ml it would be: OD260 x 50 ug/ml x dilution factor)
How would you prepare a sample to measure in the spectrophotometer with a dilution factor of 10, using a total volume of 100 ul?
10 ul sample + 90 ul H2O
1 part sample: 10 parts total
DF = 10 (total parts/sample parts)
How would you prepare a sample to measure in the spectrophotometer with a dilution factor of 100, using a total volume of 100 ul?
1 ul part sample + 99 ul H2O
1:100 ratio
DF = 100
_____ plasmid of super high quality will be supercoiled and will run _____ than its expected size.
Uncut; faster
Partially cut plasmid in an _______ runs _____ and may be bigger in size
open circle conformation; slower
_____ DNA runs true to size.
linear
If you add 4 ul of plasmid miniprep to 96 ul of H2O, what is the dilution factor?
4 parts sample : 100 parts total
DF = (100 parts total / 4 parts sample)
DF = 25
What is the significance of an A260/A280 ratio of less than 1.8?
There is contamination with proteins which makes the ratio smaller
There may be potential problems going forward with sequencing since the sample is not pure
What is the significance of an A260/A280 ratio greater than 2.0?
Not necessarily indicative of a problem but can suggest a poor quality blank eliminating too much signal near the 280 nm wavelength
This could also indicate that there is contamination of RNA in the sample extraction (observed if RNAse is not applied during DNA extraction)
You measured an undiluted miniprep sample and got A260 = 1.856 and A280 = 1.040. Is this sample pure plasmid? Why or why not? What is the DNA concentration in both ug/ml and ng/ul?
Purity = A260/A280 = 1.856/1.040= 1.78
This is essentially between 1.8 and 2.0 so it is considered pure.
1.856 x (50 ng/ul / 10 DF) = 92.8 ng/ul
Also 92.8 ug/ml
We used ______ to purify plasmid DNA away from contaminating proteins and other cellular contaminants in preparation for DNA sequencing and to concentrate our miniprep samples by precipitating and resuspending in a smaller volume.
DNA precipitation
DNA is a ___ biomolecule because of the (-) phosphate groups
polar
DNA is _____ in water.
soluble
(Readily reacts with water)
A DNA precipitation protocol results in the DNA becoming
insoluble in water
(The DNA becomes white, stringy, and falls out of solution)
What is the theory behind DNA precipitation?
To make the DNA insoluble by adding positive (+) ions to neutralize the negatively (-) charged phosphate groups of the DNA backbone
What is the potential problem with DNA precipitation?
Water is extremely polar → prevents the positive ions from interacting with the DNA
(Will interact with the salts just as well as the DNA molecules
If we just throw salt in, the water molecules will sequester the ions and not interact with DNA.)
What is the solution to this problem?
To add an alcohol that is less polar than water which allows positive ions to mask the charges on the DNA backbone
What alcohols can be used in DNA precipitation?
100% isopropanol or 100% ethanol
Efficiency is thought to improve by keeping the solution ____.
ice cold
________ is used to wash away residual salt.
70% ethanol
(30% water is just enough water to dissolve the salts.
So that when we pour the supernatant off, we dissolve the salt)
What are the pros and cons to using 100% isopropanol?
Pro: requires less total isopropanol which allows you to work in smaller quantities
Cons: pellet is more prone to dislodging from the tube, precipitates more salt, and takes longer for all residual alcohol to evaporate
______ is a highly purified polysaccharide derived from oysters.
Glycogen
(Glycogen molecules are highly branched structures composed of thousands of glucose molecules bonded to each other)
Glycogen is an inert carrier. What does this mean?
It is free of host DNA/RNA
Glycogen is _____ in ethanol solution.
insoluble
(In the presence of salts, it forms a precipitate that traps the target nucleic acids
During centrifugation, a visible pellet is formed which greatly facilitates the handling of target nucleic acids)
What is the purpose of using glycogen?
It makes the pellet more visible and in the process of forming the precipitate, it is trapping the nucleic acids
______helps the precipitates crystallize and form more efficiently
Cold temperature.
What is the DNA precipitation protocol?
Add 1/10x volume of 3M sodium acetate
Add 1 ul of 20 mg/ml glycogen
Add 2.5x volume of 95-100% ethanol
Centrifuge and remove supernatant
Wash with 1 ml of 70% ethanol
Centrifuge and remove supernatant
Resuspend pellet in the appropriate volume of ddH2O
What is needed for Sanger Chain Termination?
-DNA template to be sequenced
-Complementary primer (oligo)
(Single primer - only making single stranded DNA)
-DNA polymerase (Klenow fragment)
-Radioactive label
-Excess of normal deoxynucleotides (dNTPs)
-Limiting amounts of dideoxynucleotides (ddNTPs) aka chain terminator
Deoxynucleotides (dNTPs) lack a _______ and are the nucleotides normally used by DNA polymerases during DNA replication.
2’-hydroxyl group
Dideoxynucleotides (ddNTPs) lack ________, they can be used as substrates by most DNA polymerases.
both 2’ and 3’-hydroxyl groups
Once the ddNTP becomes incorporated into the growing chain, additional nucleotides can not be added because of the absence of a 3’-hydroxyl group. The ddNTP, therefore, causes the ______.
synthesized DNA chain to terminate
In automated DNA sequencing, labels are ______.
flurorescent
(NOT radioactive)
In current automated methods of sequencing, _________ is used instead of the four independent termination reactions.
one combined reaction
(This is accomplished by using labels specific for each ddNTP. In other words, each of the chain terminators is tagged with a unique fluorescent dye.)
The population of molecules resulting from the mixture of chain terminators is separated by size on a denaturing high- resolution ________ gel via capillary gel electrophoresis.
polyacrylamide
(Much finer pores that differentiate single nucleotide differences)
We are using _______ for our DNA sequencing service
Genewiz
The _____ indicates which terminator caused termination at that position in the sequence.
dye
_______ is the process of working out the order of the bases in a strand of DNA.
DNA sequencing
What are the steps to DNA sequencing?
To start the sequencing reaction, everything is heated to 96℃ which separates the DNA into 2 single strands.
Temperature is then lowered to 50℃ which enables the DNA primers to bind to the plasmid DNA.
Temperature is then increased to 60℃ where the enzyme DNA polymerase binds to the primer DNA.
DNA polymerase adds bases until a terminator base is added.
Everything is heated to 96℃ again which separates the new DNA strand from the original strand.
Early analysis shows that you successfully cloned your 1200 bp gene of interest into the pUC19 vector, but to confirm that you do indeed have the correct gene/sequence cloned, you sequence the clone. A fellow labmate said that they consistently obtain 500 bases of good sequence with each sequencing reaction they set up. Given this information, how many primers do you need to completely sequence your gene in the forward direction only?
At least 3 primers
(Need to have overlap between sequence generated to confirm we have the entire insert sequenced)
If both RE mapping and PCR suggest the calpain ASD is present, do we still need to do sequencing? Why or why not?
Taq polymerase is error-prone
Taq polymerase was in the Gotaq green master mix
It has no proof-reading ability
It is possible that mutations got into our gene
If both RE mapping and PCR suggest the calpain ASD is present, do we still need to do sequencing? Why or why not?
Taq polymerase is error-prone
Taq polymerase was in the Gotaq green master mix
It has no proof-reading ability
It is possible that mutations got into our gene
