molecular lab final

ligation reaction contents

2x ligation buffer/atp, cut pAMP, cut pKAN, water, ligase

when the ap is incubated with an appropriate substrate

a chemical reaction takes place that deposits a colored reaction product on the membrane

recombinant dna molecule

a dna molecule with a foreign stretch of dna inserted into the original plasmid

ethidium bromide

a fluorescent dye that absorbs lights in the UV range. it requires special handling

cDNA

a molecule that has been synthesized from an RNA template. in nature, the synthesis of it occurs during replication of retroviruses and retrotransposons. during this process, a viral or transposon RNA is copied into a dsdna molecule that can then be integrated into the genomic dna of a host

how could PCR be used to distinguish between the recombinant molecule and the vector? how would you design primers that could accomplish this task?

a primer that binds to the insert would have to be used that way only the recombinant molecule would be amplified and the vector would not. then the sample is only the recombinant dna

pcr (polymerase chain reaction)

a rapid procedure for the in vitro enzymatic amplification of a segment of dna. it exploits specific features of dna replication and uses a sea polymerase that is functional and stable at high temperatures.

restriction enzymes

a standard tool used in many molecular biology labs to map dna sequences, determine the genotype go lab organisms, and assemble novel recombinant dna molecules

southern blotting

a technique for transferring dna fragments from an agarose gel to a membrane. the membrane is made of nylon or nitrocellulose. the DNA will transfer to the membrane in the same position that it held on the DNA gel. then, the DNA will be fixed to the membrane by cross linking it using uv radiation (when using nitrocellulose, the dna is fixed to the membrane by drying in a vacuum oven)

a solution that is 1 N

contains 1 gram equivalent weight of a substance in 1 L of solution

taq polymerase

functions at temperatures up to 94°C, thus making it possible to automate the in vitro amplification of dna

serological (or blow-out) pipette

has a series of calibration marks along the stem, down to the tip. using these calibration marks, the desired volume can be pulled up. the entire volume is then empties into the receiving vessel

mohr or measuring pipette

has calibration marks that do not extend to the tip. it is used like the serological pipette, except that the delivery of the desired volume requires stopping the delivery at the bottom calibration mark

volumetric delivery pipette

has one calibration mark on the stem, with and expanded portion below. this pipette delivers one specific volume. to deliver the contents of a full volumetric pipette accurately, allows the liquid to flow freely into the receiving vessel. when the flow ceases, touch the tip to the pipette to the inside of the vessel

denaturation

high temperature melts template dna

procedure overview for heart/liver lab

human tissue sample -> isolate total RNA -> synthesize cDNA using oligo dT primer and RT (RT reaction) -> PCR amplify using tissue-specific primers for TNNT2 and HPX (PCR reaction) -> analyze gene expression

mini prep can be neutralized with

ice-cold KOAc

annealing

low temperature allows primers to anneal to the complementary sequences

RT PCR reaction contents

master mix (water, buffer, dNTPs, RT), oligo dT primer, total RNA)

microcentrifuge tubes

molecular biology experiments are often set up in disposable 1.5 ml micro centrifuge tubes

molecular biologists often use plasmids as

vectors to transform bacterial cells with specific dna sequences

In our plans for constructing a recombinant molecule, we used a restriction enzyme cloning strategy. What other cloning methods could we have used to assemble our desired recombinant plasmid?

we could have used transformation

what methods could you use to test for the successful construction of the desired recombinant plasmid?

we could use antibiotic resistance; we could isolate the plasmid, use RE, and run it on a gel; we could seq the plasmid

rflp (restriction fragment length polymorphisms)

when 2 or more different dna samples are digested by the same restriction enzyme, the resulting fragments will vary in number and size between the different dna samples

transformation recovery phase

plasmid replicated and expresses antibiotic resistance genes. after, the bacteria are plated onto an antibiotic-containing culture medium.

overnight suspension cultures are used for

preparing large amounts of plasmid DNA from bacterial cells and for inoculating mid-log cultures of bacteria. It is important that these overnight cultures be started from a genetically homogeneous population of cells

squeeze the s valve

this will siphon liquid up into the pipette

southern blot procedure: B. set-up

stack from top to bottom: weight (book), stack of paper towels (dry), 2 pieces Whatman filter paper (pre-wet), nylon membrane (pre-wet), gel, 2 pieces Whatman filter paper (pre-wet), wick (pre-wet), petri dish platform, glass dish

gel electrophoresis

takes advantage of the fact that dna is negatively charged and will move in an electrical field to the positive pole

extension

temperature is raised to optimal temperature for dna polymerase activity

pcr contents

template dna, forward primer, reverse primer, MgCl2, master mix

spectrophotometry of genomic and plasmid dna

the 260/280 and 260/230 ratios are measures of nucleic acid purity. pure dna should have a 260/280 ration of 1.8. if this ratio is appreciable lower, the sample may have contaminants such as proteins or phenol. the 260/230 ration is a secondary measure of NA purity and is often higher than the 260/280 ratio. the ratio commonly falls between 1.8-2.2 for pure NA. if the 260/230 ratio is lower, there may be co-purified contaminants

to analyze which specific cDNAs are in a sample

the cDNAs are typically amplified by PCR using primers specific for the transcripts of interest. Then, the PCR products can be observed by running an agarose gel. Presence or absence of a specific amplicon can then be detected by imaging the gel. RT-PCR is a powerful method for identifying patterns of gene expression in clinical tissue samples such as tissue biopsies

the RNA-dependent dna polymerase

the core enzyme responsible for cDNA synthesis

fragment analysis using a standard curve

the distance any fragment migrates along the gel is inversely proportional to the log of its size in molecular weight. bp length can be used as a proxy for molecular weight.

pcr cycles

the dsdna is denatured, the primers are annealed, and the new cdna is extended by taq pol

Tm is determined by

the length and the G:C content of the primer

our cDNA synthesis will only produce dna copies of mRNA, not ribosomal RNA or tRNA. How have we made our cDNA synthesis reaction specific for mRNA?

the oligo dT primer is complementary to the polyA tail, which is only found on mRNA

reverse transcriptase

the product is the reverse of the expectation of the central dogma (RNA is transcribed into DNA instead of DNA being transcribed into RNA as is more common in prokaryotes and eu)

if qRT-PCR is performed,

the relative abundance (quantity) of specific RNA molecules can be calculated

during the RT reaction, a specific cDNA molecule can be synthesized only if

the specific RNA template is present

not/bcip

the substrate, produces a purple reaction product

preparing work area for working with RNA

to avoid contamination, use gloves and filter tips (prevent contaminants that are inside of our micropipettes from being blown into our reactions), decontaminate work area with a solution that inactivates RNase enzymes

squeeze the e valve

to dispense solution

why do we always present the original dna gel and the probe results together in the same figure?

to show where bands were and which ones actually had the gene of interest

purpose of vector NTI

use tools to analyze the sequence of your gene, design PCR primers that can be used to amplify its sequence, and plan the construction of a recombinant plasmid containing the gene's sequence

digital micropipettes

used to measure volumes in the microliter range, for volumes that are 1000 µl or less

RT reaction

used to synthesize cDNA for studies of gene expression patterns, for quantifying gene expression, and for molecular cloning experiments

glass pipettes

used to transfer volumes 1-25 ml. volumes larger than 25 ml are often measured using graduated cylinders.

small-range pipette

0.5-10 µl

digital micropipette rules

1) never use a pipette without a tip in place 2) never lay down a pipette that has a filled tip 3) never let the plunger snap back after withdrawing or ejecting fluid 4) never crank the volume past the indicated range 5) never pipette caustic chemicals (including acids and bases)

RT PCR steps

1. 25°C allows the oligo dT primer to anneal, 2. 45 min allows the RT to happen, 3. 85°C inactivates any remaining RT enzyme

plasmid dna isolation (alkaline lysis method/mini-prep)

1. add overnight e. coli suspension to tube 2. pellet and decant- spin, pour off supernatant 3. wash- add ste (sodium chloride/tris/edta), resuspend, spin, pour off supernatant 4. resuspend- add cold gte (glucose/tris/edta), resuspend 5. incubate at room temp 6. lyse cells- add ads/naoh 7. incubate on ice- the solution will become relatively clear as the ads lyses the cells and naoh denatures the dna 8. neutralize and reanneal- add cold KOAc pH 4.8 (lowers the pH, allowing the plasmid dna to reanneal) 9. incubate on ice- white flocculent material (fluffy) will appear as the KOAc precipitates proteins from the mixture. as this happens, high mw dna (chromosomal dna) becomes entrapped in the precipitate) 10. pellet the precipitate- spin to collect the precipitate along the side of the tube 11. transfer supernatant to new tubes- the supernatant contains low mw proteins and nucleic acids 12. precipitate the plasmid dna- add isopropanol, invert, let stand 2 minutes. isopropanol precipitates nucleic acids quite rapidly. however, after some time it will also begin to precipitate proteins 13. pellet plasmid dna immediately- spin 5 min to pellet NA 14. decant the isopropanol- pour off the supernatant 15. wash the pellet- add 70% ethanol, flick (the dna pellet is not soluble in the ethanol, so it will not resuspend during the washing) 16. pellet plasmid dna- spin 17. decant the ethanol- pour off supernatant from both tubes. 18. remove all ethanol- evaporate remaining ethanol in speed vac for 7 min 19. resuspend in te buffer- add te (tris/edta) buffer

dna labeling procedure

1. add template dna to water 2. heat denature the dna in boiling water for 10 min and ice for 30 sec 3. add dig-high prime to denatured dna. it contains reaction buffer, hexanucleotide mixture, dntp labeling mixture, klenow enzyme, water 4. incubate at 37°C 5. stop the reaction with edta 6. assess the degree of label and add dig easy hybridization solution

southern blot procedure: A. denature the fragments

1. after running the gel, cut it in half and save the portion that contains the size markers, unknown plasmid, and genomic lanes. 2. measure the length and width of the gel 3. incubate for 15 min with denaturing solution (0.5 M naoh, 1.5 M nacl). this causes dsdna to denature into ssdna 4. place in neutralizing solution (1.0 M tris, pH 8.0, 1.5 M Nacl) 5. incubate 15 min

genomic dna isolation (phenol-chloform extraction method)

1. collect the cells- pipette slurry into eppendorf tubes that contain glass beads (the glass beads will help to break open the cells and release their contents) 2. pellet the cells- centrifuge 16,000 x g/1 min 3. remove the supernatant, resuspend in 400 µl of te buffer 4. lyse the cells- add sds (ads is a detergent that will dissolve the lipid bilayer of cells) 5. mechanical lysis and dna purification- add phenol and chloroform 6. vortex-vortex, ice, vortex 7. phase separation 1- spin 15 min at 16,000xg, makes aqueous and organic layer 8. purification- save the top aqueous layer (contains dna), add 450 µl chloroform 9. phase sep 2- spin 15 min 10. dna precipitation- save aqueous layer, add 100% ethanol, freezer for 30 min 11. pellet precipitated dna- spin 15 min 12. pour off supernatant while being careful not to disturb pellet 13. wash dna pellet- add 70% ethanol- this ethanol was contains enough ethanol to keep the dna insoluble while washing away and aq contaminants , spin 5 min 14. ethanol evaporation- pour off supernatant, dry off ethanol with air manifold 15. rehydrate dna- add the buffer

cloning procedure overview

1. digest pAMP and pKAN with BamHI and HindIII 2. check digestion progress on a gel 3. perform ligation reaction

ligation procedure

1. heat kill restriction enzymes 2. place on ice 3. add ligation reagents 4. incubate

basic biosafety rules

1. keep bench tops free of all non-essential materials. bags and coats can be stored under the lab benches or on the front table 2. wear suitable clothing and footwear. closed-toed shoes must be worn at all times 3. eating, drinking, smoking, storing food, applying cosmetics, inserting or removing contact lenses are not permitted 4. use appropriate pipetting devices 5. tie back or restrain long hair 6. decontaminate and disinfect work surfaces before and after working with live microbes 7. disinfect all materials that come in contact with bacteria. Pipettes and micropipette tips should be placed in disinfectant immediately after use. Never lay these down on the bench tip 8. use aseptic (sterile) techniques when handling microbial cultures 9. perform all procedures in a manner that minimizes the creation of aerosols 10. wash your hands before leaving the lab and at any time after handling materials known or suspected to be contaminated

pre hybridization procedure

1. place the southern blot in a seal a meal bag and heat seal all but one edge 2. add 5 ml of pre hybridization solution and heat seal the final edge, being careful to remove as much air as possible 3. incubate with shaking for 2 hrs at 42°C

hybridization of labeled probe to target dna procedure

1. pour off prehybridization solution 2. place blots in the hybridization cylinder dna- side up. add denatured probe, cap canister. put in oven

colorimetric detection of probe binding procedure

1. rinse membrane 2. block membrane- incubate in blocking solution 3. incubate membrane with antibody- add antibody conjugate 4. wash 1: wash buffer 5. wash 2: wash buffer 6. equilibrate the membrane- transfer to detection buffer 7. add substrate- add nbt/bcip 8. incubate in the dark 9. stop the reaction- wash with di h2o

post-hybridization membrane washes procedure

1. save probe solution 2. wash membrane in 2x ssc 3. wash membrane in 0.5x ssc 4. air dry filters

colorimetric assay steps

1. synthesize dig-labeled probe against sequence of interest 2. probe binds to dna template 3. antibody binds to dig 4. ap reacts with not/bcip substrate 5. detect purple reaction product on blot 6. analyze results

post- southern- blotting procedure: dna fixation

1. take apart the blot layer by layer until you get to the membrane 2. before removing the membrane, mark the position of the wells and the left and right corners of the blot with a ball point pen 3. remove the membrane and rinse in 5x sac buffer for 1 min at room temp. this will remove the gel debris and particulate contaminants that were introduced during transfer 4. disassemble the remainder of the stack and rinse the dish and support with warm water to remove salt 5. affix the dna to the membrane by exposing the membrane for 3 min in the uv crosslinker 6. the mem can be stored dry until the dna-dna hybridization steps are carried out

lab #2 objectives

1. to digest lambda phage genomic DNA with 3 different restriction endonuclease, 2. to use a standard curve to quantitatively demonstrate that different restriction enzymes cut at different sits in the same dna molecule and thus produce different patterns of bands on a gel

medium-range pipette

10-100 µl

large-range pipette

100-1000 µl

the gel matrix

acts like a molecular sieve by sorting the fragments based on size

what selection markers are available for your desired recombinant plasmid?

ampicillin

RT-PCR and qRT-PCR combine cDNA synthesis and PCR techniques to

amplify DNA copies of RNA molecules that are present in a sample. These methods use 2 main steps: 1. RNA molecules are used as a template and copied into cDNA molecules by RT enzyme, 2. the resulting cDNA molecules are used as a template for PCR amplification

both primers in a PCR experiment must have

an appropriate melting temperature (Tm) so that they will denature from the template during the melting step of the PCR cycle, but also anneal quickly with the template during the annealing step and remain annealed during the extension step

to synthesize cDNA from the mRNA transcripts present in each total sample we will use

an oligo dT primer that complements the polyA tail of mRNA. this will ensure that mRNA template is copied by the reverse transcriptase, rather than rRNA or tRNA, as these RNAs do not have the extensive polyA sequence present in mRNA

when a bacterium has taken up a new plasmid it is said to

be transformed

cDNA produced by RT is typically further amplified

by PCR using primers specific to the transcript of interest

how can one detect which bands not he membrane contain the gene of interest?

by performing dna-dna hybridization using a probe that will hybridize specifically with the gene of interest

RT-PCR

cDNA synthesis followed by PCR

endonuclease

cleave nucleic acids at internal positions in the molecule by recognizing a specific sequence of nucleotides and then catalyzing the hydrolysis of phosphodiester linkages in each strand of the dna molecule

dna oligonucleotide primer

complementary to a short segment of the template dna. since both dna strands in a ds sample can serve as templates for dna synthesis, this primer is supplied for each strand (sense/antisense or forward/reverse)

vector nti: add restriction enzyme cut sites to out gene to introduce sticky ends. these must be

cut sites for a restriction enzyme that is not already present in the gene (non-cutter)

prior to hybridizing the dna probe to the membrane, the probe is labeled with

digoxigenin (dig). after hybridization, the dig is recognized by an anti-dig antibody. the antibody is conjugated with alkaline phosphatase (ap)

dna digested using RE produces

dna fragments that can be separated based on their molecular weight using gel electrophoresis

what happens during a transformation

dna molecules pass through one of several hundred pored in the bacterial cell wall. pores are formed by fusion of the outer and inner cell membrane of e. coli in zones of adhesion. pores are created when e. coli is exposed to cold CaCl2. CaCl2 crystallizes the fluid cell membrane, thus stabilizing the distribution of charged phosphates of the phospholipids. Ca2+ shields the negative charges of the exposed phosphate groups of dna and promote binding of the dna to the cell membrane. heat shock generates a thermal gradient that sweeps the shielded dna through the pores and into the bacterium

dna-dna hybridization

done by generating a dna probe that is complementary to the sequence of interest. the probe will hybridize specifically to the sequence of interest. the probe will be labeled with a marker that we can visualize. the labeled probe allows us to visualize the bands that contain the sequence of interest. by comparing the labeled bands on the membrane to the original dna gel image (which shows all the restrictions fragments and the dna size markers), we can determine which dna fragments contain our sequence of interest

squeeze the A valve and simultaneously squeeze the bulb to

expel air

the desired sequence called the

insert is ligated to the plasmid using dna ligase

colorimetric assay

involves a color reaction that takes place on the membrane specifically where the dna probe has hybridized to its complement. this will allow us to visually detect which specific dna fragments contain the gene

the streak-plate method is used to

isolate single bacterial cells from one another

primer dimers

occur when primers anneal to incorrect dna sequences. usually when 2 primers anneal to each other and produce short amplicons. appear on a gel as a low mw (<50 bp)

pipetting bulbs

often used with glass pipettes to pull up and expel liquids. a commonly-used type of pipetting bulb has 3 valves that are used to 1) release air from the bulb 2) to fill the pipette with liquid, 3) to expel liquid from the pipette

since RNA transcripts are single stranded it only requires

one primer for the RT reaction. this is distinct from a PCR reaction in which ds template is copied by a pair of primers

selective medium

only the cells that can survive are transformed cells that have express antibiotic resistance proteins. untransformed cells are killed.

master mix contents

reaction buffer, taq pol, dNTPs

the nylon membrane will provide a

robust support for the dna being studied. this will allow us to probe the fragments for the gene of interest, using dna-dna hybridization

the mini prep solution will become relatively clear as the

sds/naoh lyses the cells and dissolves the organic material

plasmids

small circular dna in bacteria. genes that confer resistance to specific antibiotics