Exam 2 Flashcards
how to identify the fragment of interest in a DNA gel extraction
Place gel on a light box appropriate for the dye used. Avoid exposing DNA to mutagenic radiation for longer than necessary (UV for EtBr).
how to isolate a DNA band from a gel
Physically remove DNA band of interest with a razor blade. minimize the amount of gel around the band that is excised
how to clean up an isolated DNA band via electroelution
recover DNA fragments from a particular region of agarose or polyacrylamide gel. The gel piece is placed in a dialysis bag with buffer. Electrophoresis causes DNA to migrate out of the gel into the dialysis bag buffer. Recover fragments and purify using phenol/chloroform extraction followed by ethanol precipitation
simple, rapid, high yield method
how to clean up isolated DNA fragment via spin column extraction
Dissolution of gel slice at 50 C. DNA adsorption on silica membrane or beads in high salt buffer. Ethanol washes salt and impurities out of column, DNA remains bound. Low salt solution or pure water displaces DNA (elution)
how to clean up an isolated DNA fragment via enzymatic method
Agarase enzyme digests agarose polysaccharide core. Use low melting point agarose to make the gel so you can melt gel at 70 C. Then transfer to 45 C and add enzyme.
DNA can be used directly in many applications without need for further purification
other methods of clean up after gel extraction
E-Gel CloneWell: gel has two rows of wells. insert sample in top well, run until desired fragment is in bottom well, remove from bottom well.
Automated Gel Purification: machine does a very precise extraction. expensive
why is it important to avoid excessive UV exposure when visualizing a gel?
DNA is damaged rapidly by UV radiation. EtBr dye is visualized under a UV light, but exposure for over 30 seconds fatally damages most DNA. SYBRSafe is a good alternative to EtBr because it is visualized under blue light which is not harmful at all.
biological functions of ligases
Lagging strand DNA synthesis
genetic recombination
DNA repair
Ligation reaction components
Buffer: ATP (energy, don't freeze-thaw too much!), DTT (antioxidant), BSA (stabilizer), Mg2+ (enzyme cofactor) Plasmid Insert DNase-free water T4 DNA Ligase (packaged in 50% glycerol)
explain ligation in sticky vs blunt ends
sticky ends: have complementary overhangs which make ligation efficient
blunt ends: no overhangs or complementarity. ligation is inefficient and needs high concentration of ligase and DNA. molecular crowding agents like PEG 8000 can improve intermolecular ligation
intra- vs inter- molecular ligation
intramolecular ligation: within the same molecule (circularization). favored by LOW concentrations of ends. length of molecule is inversely proportional to frequency of ligation
intermolecular ligation: occur between different molecules. dependent on concentration of ends (not plasmid:insert ratio). favored by INCREASE in concentration of ends
explain the results of different concentrations of ends and insert:vector ratios
Very high end concentrations causes concatamers. If insert:vector ratio is high, concatamers of inserts form and vice versa, equal ratio forms plasmid insert concatamer
Very low end concentrations has bad success rate. If high i:v ratio, few colonies have plasmid and none have insert. If i:v low, lots of colonies with plasmid but no insert. If equal, some colonies with plasmid but not insert
Medium end concentration with high i:v has highest product yield when plasmid is dephosphorylated. With low i:v plasmid dimers or recircularizep plasmids form. With equal i:v there’s a 50% chance of correct ligation and highest yield of product
What is Calf Intestinal Alkaline Phosphatase
CIP dephosphorylates 5’ phosphate groups from DNA, RNA, and ribo/deoxyribonucleoside triphosphates. Effect is that DNA cannot self ligate after CIP treatment. This reduces vector background noise and favors clone insert
replaces phosphate with OH group, no phosphodiester linkage can form with self. insert is still phosphorylated so it can form linkage with plasmid (result NICKED circle)
How to calculate ligation reaction concentrations and volumes
i = ratio (insert length bp / plasmid length bp) p
This equation gives an answer of insert = #plasmid. Then you can substitute this equation for i in the equation
i + p = total DNA needed (calculated from reaction volume x total DNA concentration)
to get #p + p = total DNA
solve for p to get ng of plasmid needed. use p to solve for ng of insert needed.
finally, use given plasmid and insert concentrations to calculate volumes in µl
how to use linkers in cloning
use ligase to add linker molecules (containing restriction sites) to your DNA insert. then cut at the restriction sites and insert into a vector with complementary overhangs.
how to use adaptors in cloning
ligate double stranded cDNA with adaptor molecule (short sequence with one blunt end and one sticky end lacking the phosphate) so the blunt ends join together. remove unligated stuff and dimer adaptors. phosphorylate the adaptor-ligated cDNA with a kinase. ligate cDNA to a vector with an overhang complementary to the adaptor
advantage is you do not need to treat the adaptor modified DNA with restriction enzyme
explain genomic DNA libraries
how to make: isolate DNA from cells. digest with REs, nucleases, or mechanically. ligate into vectors and insert into bacteria to amplify. isolate the DNA to form a collection of genomic DNA library.
potentially includes entire genome of the organism and is large and complex. expression is difficult due to introns and possible lack of entire gene. can use any vector type. good to pull out non-expressed sequences (promoters, intons, etc)
explain cDNA libaries
how to make: isolate mRNA and reverse transcribe into cDNA. digest with REs and ligate into vectors. insert into bacteria for amplification and then isolate the DNA for a collection of cDNA library.
includes only the mature mRNA sequences of genes expressed in source tissue. fewer clones, but only the most important sequences. proteins can be readily expressed. clones are small, so use vectors like lambda, plasmid, or cosmid/fosmid.
types of polymerase for PCR
non-proofreading:
Taq. lacks 3 - 5 exonuclease activity. results in a single nucleotide overhang, usually A, which can be cloned into T vectors.
proofreading:
results in blunt ends and must be cloned into a blunt ended vector or needs a single A overhang to ligate into a T vector
advantages and disadvantages of TA cloning
utilizes the unpaired A residue produced by non proofreading DNA polymerase to pair with a linear T vector
Advantage: universal, speed, lack of dependence on REs
Disadvantage: no directionality. must pick and analyze several closes when orientation is needed
how to make a T vector
- digest a vector with a restriction enzyme that generates an unpaired 3’ T residue
- use terminal transferase and ddTTP to add a single T residue to the 3’ termini of vector
- use template independent terminal transferase activity of Taq polymerase to add a T residue to the terminal 3’ end. if only dTTP is present, it will add it instead of an A residue
- purchase a ready made T vector
describe TOPO TA cloning
utilizes topoisomerase of vaccinia virus which relaxes DNA supercoils by transient breaks in one DNA strand. cleavage occurs with high specificity. nicking of the DNA occurs as a result. Because of conserved bond energy, the reaction is readily reversible and the phosphotyrosyl-DNA intermediate can:
catalyze reformation of the original duplex or
create a recombinant DNA by forming a phosphodiester bond
how to clone blunt ends made by proofreading polymerases
TOPO blunt-end cloning. Same process as TOPO TA cloning. The topoisomerase cleaves and then allows for new bond formation. primers are made with no 5’ phosphate so if plasmid is also lacking the 5’ phosphate nothing will happen, make sure to order primer with 5’ phosphate or add one
describe In-Fusion (Clontech) cloning
a recombination-mediated technique for cloning any PCR product into any vector. design primers with an extra 15 bp at the 5’ end that matches the position in the vector where you want to clone the PCR product. Both ends of the PCR product need the matching sequence. With the added vector complement sequence, it’s easy to get the desired construct in a reaction with the In-Fusion enzyme.
Can be used to clone in multiple fragments at the same time
how to fix reading frame problems via PCR
carefully design PCR primers to yield desired RF. plan cloning strategy from the beginning for desired clone. Add or subtract nucleotides from 5’ ends of primers. instead of cutting gene out with REs, amplify by PCR and add RE sites to primers for cloning.
explain ampicillin and resistance
ampicillin is a B-lactam antibiotic that interferes with bacterial cell wall synthesis (B-lactam is part of cell wall)
B-lactamase is an enzyme that breaks the B-lactam ring of the antibiotic and nullifies the antibiotics effects
explain kanamycin
interact with 30S subunit of bacterial ribosomes and induces high levels of mistranslation, indirectly inhibiting translocation during protein synthesis
define transformation
the genetic alteration of a cell (bacteria) caused by the uptake of foreign DNA
what is the barrier that must be overcome for transformation occur
the hydrophobic interior of the phospholipid bilayer of cell membranes prevent polar molecules (DNA) from passing through the membrane. We need a way to get our foreign DNA inside the cell (heat shock/chemical or electroporation)
how to make cells chemically competent
- wash in NaCl - remove ions and contaminants
- CaCl2 added - makes membranes more permeable
- Glycerol added - allows cells to be frozen, preserved in cryostatic state
can also buy competent cells
Heat shock procedure
Keep cells cold! Don’t mix vigorously (even pipetting up and down too much)!
Chill cells on ice (DNA binds to cells)
Heat to 42 C to make membrane fluid, porous, allowing DNA into cell
Chill on ice again
Add SOC medium with no antibiotic. grow 30-60 minutes. gives cells time to grow and produce resistance proteins
Plate on selective media plate
electroporation procedure
Close 1st switch: capacitor charges up and stores a high voltage
Close 2nd switch: voltage is discharged through cell suspension liquid
A typical pulse is 10,000 to 100,000 V/cm lasting a few microseconds to a millisecond
Temporary aqueous pores are formed by the disturbance of the phospholipid bilayer caused by the electric shock and charged DNA molecules are driven across the membrane by increase in electrical potential of the cell membrane
Review the lac operon transcription mech
LacI gene encodes a repressor. If no lactose is present, the repressor will bind the lacZ operator and block transcription. If lactose is present, allolactose will bind the repressor and stop it from blocking the operator. Transcription occurs and B-Galactosidase, permease, and transacetylase are made.
