Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
76 Cards in this Set
- Front
- Back
|
Difficulties working with RNA |
Complicated by Ribonuclease enzymes in cells and tissue, which rapidly degrade RNA -these Ribonuclease can remain active on lab glassware, water, and disposables |
|
|
RNA extraction |
Guanidinium thiocynate-phenol-chloroform extraction *more complicated than DNA extraction *add cell homogenate (lyzed cells) to centrifuge tube *add to this aqueous sample water saturated phenol, cholorform, and denaturing solution (guanidium thiocynate) *results in upper aqueous phase that contains nucleic acids and lower organic phase (mainly chloroform) which contains proteins **RNA is recovered from the aqueous phase by precipitation with Lithium Chloride |
|
|
Chemicals for RNA isolation and their functions |
*Phenol and chloroform *Guanidinium Thiocyanate *Lithium Chloride |
|
|
Phenol & Chloroform |
Allow nucleic acids to separate from proteins but are both hazardous/toxic and extraction is time consuming |
|
|
Guanidinium Thiocynate |
Denatures proteins, including RNases, and separates rRNA from ribosomes |
|
|
Lithium Chloride |
Will precipitate RNA but not DNA |
|
|
RNA Lab precautions |
*use only unopened bags of tubes *must order special RNAse-free tubes and tips *Use dedicated pipettors *Fresh gloves that have not touched anything else in lab *DEPC *RNase-free water |
|
|
What is DEPC? |
*Diethylpyrocarbonate(DEPC): used in laboratory to inactivate RNase enzymes in water and on laboratory utensils. It does so by the covalent modification of histidine residues |
|
|
What is RNase-free water used for? |
Used in the handling of RNA in laboratory, to reduce the risk of RNA being degraded by RNase |
|
|
Northern Blots |
*Similar to Southern except now using RNA *No need for restriction digestion of samples because RNAs are already small fragments *must work in RNase free environment |
|
|
What is Antisense RNA |
Single-stranded RNA that is complementary to messenger RNA (mRNA). Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and inhibiting it's translation. |
|
|
Southern Blotting Nuclei Isolation |
*Disrupt plasma membrane without disrupting nuclear envelop *nuclear Lysis buffer *must be done with cold reagents |
|
|
First step in southern nuclei isolation after tissue disrupted |
*centrifuge for to pellet nuclei *pellet is mostly nuclei *discard supernate, save pellet *pellet is mostly nuclei *NP-40 Lysis buffer |
|
|
Second step Southern nuclei isolation |
*treat with SDS and incubate *nuclei breaking open *after incubation DNA and proteins in solution *ammonium acetate removes proteins *precipitate DNA with cold alcohol *spool out |
|
|
Southern DNA quantification |
*using spec |
|
|
What do you dissolve DNA in to quantify |
TE buffer (Tris-EDTA) |
|
|
Purity check method |
260/280 absorbency check |
|
|
What specific wavelength is used when measuring DNA absorbency during DNA quantification? |
260nm |
|
|
What conversation factor from absorbance (OD) to concentration of dsDNA |
OD 1=50ug/mL |
|
|
You diluted your sample 1:40 and the absorbance at the appropriate wavelength for DNA was 0.4. What is the concentration of the original sample in mg/mL ? |
0.4 x 50ug/mL x 40 = 800 ug/mL Wavelength x 50 x dilution factor = con. Convert to mg |
|
|
What 2 detergents did we added to nuclei in order to break open the membrane and release what is on the nucleus? |
NP-40 and SDS |
|
|
Lysis buffer was added to the calf thymus when grinding then we centrifuged this. After centrifugation what was in the supernatant and what was in the pellet: |
Nuclei in pellet Cell Debris in supernatant |
|
|
What does 260/280 tell is about our sample? |
DNA purity to determine of protein contamination. 260nm: max DNA absorbance 280nm:max protein absorbance |
|
|
Southern DNA restriction digestion |
*Use restriction enzyme to cut DNA into fragments -GAATTC is recognition site - this site flanks VNTR in cow DNA - This VNTR is our target - Southern boring will be used to detect this target |
|
|
Why using EcoR1 |
*Will digest genomic DNA into thousands of different size fragments |
|
|
How is Southern different from Western? |
*Not using antibodies *Not using PAG *Target is not a protein |
|
|
How to concentrate DNA? |
*Microcon spin devices allow us to do this *centrifugation is used *special membrane in device excludes molecules of certain size (IE DNA) excludes molecules of certain size (IE DNA) |
|
|
Why Concentrate? |
Need concentrated DNA for restriction digestion in order to have enough target DNA after transfer onto membrane |
|
|
What is run on gel for digest? |
*1.2% agarose (resolves smaller DNA) *run samples: -standard 200bp ladder - + control (ecor1 cut cow DNA-pre digested) - Negative control -digested sample 1 - digested sample 2 |
|
|
Why denature gel prior to transfer? |
* ssDNA is necessary for Probe to bind *use NaOH to denature gel |
|
|
Neutralizing gel why? |
Get pH back to metal proud to transferring DNA to membrane *use Tris-HCl to neutralise |
|
|
How is blotting different from southern to western? |
Transfer buffer is different *western : 25mM This, 190mM glycine, 20% methanol *southern: 0.5 M NaCl, 10mM Tris @ pH 8.0 **the thing being transferred is different **the gel is different **the probe will be different |
|
|
Next step in southern after blotting? |
Probe hybridization |
|
|
How prepare southern blot membrane for hybridization ? |
Blocking *blocking buffer with milk protein to bind to filter and occupy areas on the blot not occupied by DNA *blocks empty sites from reacting with probe |
|
|
Why do we treat gel with NaOH before blotting? And why is this important? |
Denature: to make sure DNA is single stranded. *important so probe will bind |
|
|
What does hybridization buffer do? |
Treat membrane to equilibrates the membrane and prepare for probe |
|
|
Why do we incubate the membrane at 65C (what is going on during this hybridization step) what would happen if incubated at room temp? |
The probe is linking up with target sequence. If room temp probe would bind nonspecifically to other parts of DNA as well as target |
|
|
What is probe abd what is attached to it? Why important? |
*Probe is ssDNA complimentary to target sequence. *Biotin is attached *Biotin will react with Avidin on enzyme in order to highlight target sequence during colormetric steps |
|
|
What is attached to enzyme (peroxidase) so that it binds to target? |
Avidin |
|
|
At the very end of Southern we hope to see a color appear on our membrane where our target DNA is on membrane. What is the name of the substance that changes the color as a result of the enzymatic reaction? |
Choloranapthanol (purple color) |
|
|
What are the 2 blocking agents that we used and explain what each one is blocking? |
Milk protein: blocking unoccupied probe DNA sites Gelatin : blocks unoccupied enzyme sites |
|
|
What is DNA sequencing |
The process of determining the nucleotide sequence of a single strand of DNA |
|
|
Overview of DNA sequencing |
*amplify DNA target to be sequenced *make DNA single stranded *choose target strand *design primer to anneal to target strand *label primer (or label dNTPs) *Run sequencing reactions *electrophoresis of results (PAGE) *analyse electrophoresis bands |
|
|
Manual Dideoxy DNA sequencing |
*enzymatic mediated DNA sequencing *using unique property of modified nucleotide *Dideoxynucleotides are used -missing 3' OH group on the sugar |
|
|
What happens when a ddNTP is incorporated into the DNA strand that is being made during a sequencing reaction? Why? |
Lack of 3' OH group. Chain syn stops |
|
|
What is usually labeled with radioactivity in manual sequencing and what is labeled with florescent dyes in the sequencing method that uses automated analysis? |
ddNTPs - automated Primer -classical |
|
|
How many reaction tubes do we set up for one sample in classical(manual) radioactive DNA sequencing reactions? How do they differ from each other? |
4 tubes ddATP, ddGTP, ddTTP, ddCTP. They differ by ddNTP (Dideoxynucleotide) being highlighted |
|
|
How is newly sequenced DNA visualized after it has been run on gel and processed (give correct names for the output for classical manual. Method vs. Automated method and explain what they look like) |
Manual radioactive method is set up 5' to 3' with lanes labeled GATC. Bands correspond in position with which nucleotide they are (autoradiograph) Automated method looks like a flow chart kinda like a EKG (chromatograph) |
|
|
Traditional radioactive method... Sequence map |
If given template strand... Make sure it would be opposite of it |
|
|
GFP |
Green Fluorescent Protein |
|
|
What is GFP isolated from? |
Jellyfish Aequorea victoria |
|
|
What makes jellyfish glow? |
Calcium ions : light switch turns on in presence of ions and calcium ion is one of natures favorite messengers |
|
|
What is transformation? |
Uptake of foreign DNA.. Often a plasmid |
|
|
What is a plasmid? |
Originally evolved by bacteria Circular autonomously replicating DNA May express antibiotic resistance gene or be modified to express proteins of interest |
|
|
Genes on the pGlo plasmid |
*bla-Beta Lactamase -Ampicillin resistance the beta Lactamase protein is produced and secreted by bacteria that contain the plasmid. Inactivates Ampicillin
*GFP-green fluorescent protein
*araC -regulator protein |
|
|
pGlo plasmid diagram |
|
|
|
What does Arabinose operon do? |
Regulates GFP Gene in the pGlo plasmid |
|
|
What is Arabinose? |
*Source of energy and carbon for bacteria. Found in plants and normally used as food source *bacterial genes that make digestive enzymes to break down food are NOT expressed if Arabinose is absent *when Arabinose is present genes turn on. When absent genes turn off *Arabinose initiates transcription of these genes by promoting the binding of polymerase. |
|
|
What is LB agar? |
Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and Gene expression |
|
|
Methods of transformation |
*Electroporation- electric shock makes cells membrane permeable to DNA *Calcium Chloride / Heat Shock-chemically-competent cells uptake DNA after heat shock |
|
|
After growing a single glowing colony in LB broth for 1-2 days, they were pelleted by centrifugation. We then added TE buffer to resuspend pellet. What did we add after to these cells? |
Lysozyme then into freezer. To disrupt cell walls. Release of soluble components, including GFP |
|
|
What is the name of type of chromotography we used to isolate GFP? Explain how the resin (column material) attracts protein... |
HIC: Hydrophobic Interaction Chromotography sample loaded with salt water. Beads attract hydrophobic protein... Making them stick to beads in column. When salt is removed... The protein drip out into bottom column. So separates proteins. |
|
|
What is the purpose of the binding buffer rinse? Why did we do it? (was it high, medium, or low salt content) what is eluted from the column with this buffer? |
High salt buffer... Supernatent containing GFP has same salt concentration add equilibrated column. Bind GFP to column |
|
|
What is purpose of wash buffer rinse? Why we do it? High or low salt content? What is eluted? |
Low salt wash buffer. ...used to wash weakly associated proteins from column. GFP remain bound. Ring of GFP begins to penetrate upper surface of matrix |
|
|
Purpose of elution buffer? Low or high salt content? What is eluted from column. |
No salt buffer.... Used to wash GFP from the column. Ring of GFP under UV light |
|
|
What are Cell cultures used for? |
Cells services from living tissues *normal *embryonic *cancerous Transformed cells *cell lines that are derived from cancer or have been made to resemble cancer cells |
|
|
How to grow cells in vitro |
Cells are placed in culture flasks or petri dishes with appropriate medium |
|
|
Cell culture media |
Different types based upon how cells are being grown *DMEM -Dulbeccos Modified Eagle Media - Salts, serums additives include fetal bovine or defined calf serum |
|
|
Cell culture substrates |
Sterile plastic disposables (polystyrene) Extra cellular matrix for cells to adhere to (cells won't divide without adhesion to surface) |
|
|
Optimal pH for cell growth |
7.4 |
|
|
Mycoplasma contamination |
Mycoplasma.... A genus of bacteria that lack a cell wall. Without a cell wall, they are unaffected by common antibiotics such as penicillin..or other beta-lactam antibiotics that target cell walls |
|
|
Mycoplasma contamination |
Mycoplasma.... A genus of bacteria that lack a cell wall. Without a cell wall, they are unaffected by common antibiotics such as penicillin..or other beta-lactam antibiotics that target cell walls |
|
|
Contamination precautions |
*Laminar flow hood *antibiotics are added to medium *sterile disposable pipettes & flasks are used *aseptic technique *disinfect cabinet before and after working with cells *fresh gloves |
|
|
What are lag, log, and plates phases |
*lag phase: 2-24 hours after initial seeding of cells *log phase: exponential growth *plateau phase: as cells spread and use up nutrients, reduced growth or G0 is reached |
|
|
What is cell counting |
Sample is used to estimate concentration of cells in solution |
|
|
Cell count formula |
|
