2 Plasmids Flashcards

Question

After alkaline lysis what is done

Answer 1

Neutralize the previously high pH with potassium acetate Potassium dodecyl sulphate is not soluble The large complexes of denatured protiens DNA cell debris that are coated with SDS are precipitated The supernatant now has plasmids, soluble protiens, ribosomes, some RNA

Answer 2

RNase A RNA also get removed by being hydrolyzed in the basic conditions (high pH) And RNA doesn’t bind to the silica columns

Answer 3

Plasmids bind the silica membrane in HIGH SALT RNA , cellular protiens, and metabolites don’t bind Low salt or water elutes the plasmids ETHANOL DOES NOT ELUTE

Answer 4

gel electrophoresis: - can find size and quantity of DNA - can observe DNA/RNA contamination Spectrophotometry: - can show quantity of DNA or RNA - can measure salt/buffer/protien contamination Fluromotery: - can find quantity of ssDNA, dsDNA, or RNA bioanalyzer: - can find size and quantity of DNA or RNA

Answer 5

Can find size and quantity of dna from 50 bp to 10kb Quantitation is possible but not as accurate as other methods Can find DNA/RNA contaminations and plasmid DNA superstructure (like relaxed or supercoil)

Answer 6

Agarose in buffer is melted DNA bind dye added (used to be ethidium bromide but now SYBR safe less mutagenic) - gel can be stained after running too Gel is poured and dna loaded into wells Electricity applied, dna move toward + electrode (run to red) because backbone - charged DNA binding dye is visualized: - ethidium bromide reacts to UV light (but it causes DNA damage) - sybr safe reacts to blue light

Answer 7

Quality: - can show the number and relative size of dna fragments. - many bands could be expected or not (which indicates DNA/RNA contaminations) Size: - Using MW ladder to estimate DNA size - if the size is unexpected might be an unwanted DNA/RNA fragment - smaller bands could be degradation products Quantification: - compare the pixel intensity of your band to the intensity of a band of known quantity (in amount of DNA in the ladder is known - can do this using computer program or eyeball it - give an estimation

Answer 8

1. DNA size 2. Agarose concentration 3. Voltage applied 4. Buffer used 5. Presence of dna binding dye in the gel 6. Conformation of DNA

Answer 9

Size: - small faster - large slower Agarose concentration: - 0.5-2% good - high concentration let’s smaller molecules to separate better

Answer 10

Voltage applied: - dna rate of migration is proportional to the voltage applied - high voltage, lower range of separation - low voltage higher separation, but it takes longer - 3-5 V/cm of need good separation, if not use 5-8v/cm Buffer used: - buffer composition and ionic strength affects DNA mobility - TBE and TAE are the standard buffers - make sure gel and running buffer use the same buffer

Answer 11

Ethidium bromide is an intercalating agent and sybr binds to dna as well - so both slow the DNA Both dyes are also + charged, which changes CHARGE, WEIGHT, CONFROMATION, and FLEXIBILITY, of the DNA These effects also apply to the DNA ladder staining the gel after running it removes these effects BUT it takes longer

Answer 12

Order of migration slower to faster: Relaxed circular (nicked) Linear Supercoiled (intact circular) So for accurate comparison, all DNA must be linear so that shape is not a factor

Answer 13

Pros: - determines size and quantity of DNA - and quality (contaminating DNA/RNA is visible) Cons: - need high dna amount (>25ng) - Quantitation is not as accurate - no indication of any other contaminants - dsDNA only - ssDNA and RNA gels are possible but more difficult

Answer 14

Normally use nano drop Nucleic acids absorbs light at 260nm, based on this absorabnce you can find concentration using c= A/eL, contraption is in ng/microL This calculation is automated in instruments and can be modified for dsDNA, ssDNA, or RNA This method doesn’t distinguish between RNA and DNA

Answer 15

Since salts and solvent absorb at 230nm and proteins absorbs light at 280, can use ratios to find quality A260/230: - 2.0-2.2 - low ratio means contaminating reagents A260/280 - >1.8 - low ratio means contaminating protiens (or phenol)

Answer 16

Pros: - highly accurate Quantitation of DNA or RNA (>10 ng/microL) - very fast - require little sample (0.3-1microL) - a quantitative measure of quality (show salt/biffer/protien contamination) Cons: - determines presence of ALL nucleic acids (ssDNA, dsDNA, and RNA)

Answer 17

The pure sample is mixed with a fluorophore , the fluroecnsse is relative to concentration of DNA Only find quantity of DNA, not contamination (quality) Can buy fluorophore specific for ssDNA,dsDNA, and RNA , and protien The fluorophore are ORDERS OF MAGNITUDE more sensitive than absorabnce at 260 nm, which is why it can distinguish between dna rna protien

Answer 18

Thermofisher qubit machines Can tell diff between ss ds DNA , rna and protien where spectrophotometry can’t

Answer 19

Pros: - extremely sensitive (as low as 10pg/microL) - can distinguish ssDNA, dsDNA AND RNA cons: - Expensive - no measure of contamination of any kind - only finds concentration of dna not quality

Answer 20

The sample is analyzed using micro fluidic electrophoresis (Agilent) Can find quantity and quality at same time Specific to either DNA only or RNA only So can’t tell and RNA contamination if looking at DNA

Answer 21

Pros: - determines size and quantity of DNA or RNA or protien - 50bp to 12kb - can visualize DNA or RNA (not both) contamination - finds wuantity and quality at the same time Cons : - Very expensive

2 Plasmids Flashcards

(45 cards)