Projekt metode Flashcards
De mindste stykker i gelelektroforese er tæt eller væk fra brønden?
Hvorfor?
verifies the length of the produced DNA fragments
Jo længere væk fra brønd jo mindre
Negativ phosphat (negativt O)
Mest negativ
Sanger sekventering
Hvorfor ddNTPs frem dNTPs
Fordi de stopper replikation, da der ikke sider en OH men kun H, derved kan der ikke dannes phosphordiesterbindinger.
Tag polymerase
being stable at temperatures up to 95°C
afhængig af magnesium som kofaktor.
being most active in the 70-80°C, That’s hot enough that DNA won’t reanneal, but primers that have annealed at lower temperatures won’t peel off, either.
Taq polymerase works extremely well at the temperatures most standard PCR reactions require.
Fra en bakterie der lever i varme kilder
Primer design
For høj Ta resultarer i ?
For lav Ta resultarer i ?
High Ta results in primers not being able to bind to the DNA sequence
Too low Ta results in non-specific amplification where the primer binds to other positions in the sequence.
Tag polymerase cofakter.
Hvad sker der hvis Taq pol ikke har magnesium.
Hvorfor er koncentrationen vigtigt?
More critical to your PCR reactions is magnesium chloride. Magnesium is an essential cofactor for Taq polymerase3. Without it, Taq won’t be able to catalyse the addition of nucleotides to the primer or growing DNA strand.
The concentration of magnesium also matters. More magnesium added to a PCR reaction, Taq -> more active, but also less specific in polymerising only your target DNA strand.
Proteinkinase K
Funktion?
Aktive under mange forskellige x og x
Proteinase K is a subtilisin-related serine protease that hydrolyzes a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates.
Serin protease som hydrolyserer peptidbindinger.
Aktive i mange forskellige temperature og buffere
Highly characterized for more consistent performance
Optimal activity and stability for up to 24 months
Active in a wide range of temperatures and buffers with optimal activity between 20 and 60°C and a pH between 7.5 and 12.0.
No detectable endonuclease, exonuclease, DNase or RNase contaminating activities
Buffer AL
ATL buffer
Buffer AL contains the salt guanidine hydrochloride that
denatures the hydrogen bonds in water.
ATL buffer consists of sodium dodecyl sulfate (SDS),
which will denature the cell membranes.
EDTA buffer
TE formål:
TE formål: opløseliggøre dna, mens det beskytter det fra nedbrydning.
“TE” is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.
Hvorfor ikke bruge almindelig polymerase
Human polymerase har optimum omkring 37 så det kan ikke klare processen.
Det vil denature.
Agrose gel er lavet af
Hvordan kan man forme dette?
Hvordan paserer molekyler igennem.
Agarose is a component of agar. It forms a 3D gel matrix of helical agarose molecules in supercoiled bundles held by hydrogen bonds, with channels and pores through which molecules are able to pass. When heated, these hydrogen bonds break, turning the agarose to liquid and allowing it to be poured into a mold before it resets
Agrose er lavet agar. Det kan forme helix matrix som er super coiled, bundet af hydrogenbindinger. Der er kanaler og porer som molekyler kan passerer igennem.
Under opvarmning kan hydrogenbindingerne brydes, og sættes i en form.
The higher the percentage of agarose, the smaller the pore size, thus the smaller the molecules able to pass and the slower the migration.
Farve: This binds the DNA and fluoresces under UV light, allowing the DNA fragments to be visualized. The more DNA present, the brighter the band.
DNA absorbs light at wavelengths of … and proteins absorb at ? nm
DNA absorbs light at wavelengths of 260 nm,
and proteins absorb at 280 nm
Ulempe ved nanodrop
Hvilke ting kan påvirke resultatet?
Nanodrop can not determine whether or not the absorbance is measured
from DNA, RNA or protein.
Other contaminants such as salts, organic compounds, and free
nucleotides can also affect the results.
QUBIT
works by adding fluorescent dye to the testing sample, which then will attach
to selective molecules, for example, double-stranded DNA (dsDNA).
detects the intensity of fluorescence signals in the sample and calculates DNA
concentration using known standard concentrations for DNA.
measures the quantity of intact dsDNA
Hvorfor GC content?
GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding.
The G and C bases have stronger hydrogen bonding and help with the stability of the primer.
Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation.
hvad er en god længde for en primer?
Kortere primer er bedre fordi de?
Mellem 18-30 baser.
Afhænger af temperaturen.
Jo mindre primers jo bedre binder de til template.
A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.
Temp for primer
Try to make the melting temperature (Tm) of the primers between 65°C and 75°C
Tm afhænger af længden, bedre med kortere primers.
Hvordan virker Spektroskopi (nanodrop)
Hvorfor inden qubit?
Spektroskopi virker ved at sende en lys stråle med den givende bølgelængde igennem opløsningen og måle hvor stor en del af lyset der bliver absorberet.
Molekyler og atomer absorbere lys af forskellige bølgelængde baseret på deres struktur.
Vi ved dna absorberer lyset omkring 260 nm
For at tjekke om der overhoved er noget tilstede i prøven inden man laver qubit (tager lidt mere tid)
