Bio Lab Final
Transformation efficiency
# colonies (transformants)/micrograms of plasmid DNA used for transformation -5 ng pBIT DNA (positive controls) -50 ng ligated vector DNA (ligation reaction)
Calculating microliters of DNA
# ng given/concentration in microgram/mL
10X --> X Calculation
(Given X value) * ? = (new X) * (final volume)
How does a ligation reaction work?
1. Add T4DNA Ligase Buffer, PCR water, vector & insert DNA, and ligase 2. Mix solution and then microcentrifuge 3. Incubate at RT for 10 min 4. Heat inactivate reactions at 65°C for 10 min (inactivates ligase I'm guessing)
How does nucleic acid clean up kit work?
1. Binding buffer and DNA are put in spin column & micro-centrifuged DNA is in silica membrane of spin column 2. The DNA is then cleaned using wash buffer and micro-centrifuging 3. Elution buffer is added and the spin column is micro-centrifuged DNA is in the flow through REQUIRES: binding buffer, wash buffer, elution buffer, microcentrifuge tube, spin column & collection tube, & microcentrifuge
What is the function of loading dye
1. Cause DNA fragments to sink into wells 2. Enable the visualization of gel bands under UV light after gel was run
Summarize DNA cloning in 4 steps
1. Cut out desired gene with restriction enzymes (isolate insert) 2. Transfer cut out gene to the plasmid (ligation) 3. Insert plasmid into cell (transformation) 4. Grow bacteria on a plate with antibiotics (plasmid has the resistance gene)
4 ways concentrations can be stated
1. Moles/Liter (M) 2. Grams/100mL: % (weight/volume) = %grams/100mL 3. % volume (volume/volume) 4. A factor of X When making a solution, calculate each part individually & then combine them all with water to produce the desired volume of solution.
How does Sanger sequencing/chain termination method work?
1. PCR to amplify DNA 2. Add fluorescently labeled dideoxynucleotides (both 2 and 3' carbon have only H attached) 3. Gel electrophoresis 4. Use computer to analyze fluorescent labeled dideoxynucleotides at the end of fragments to determine sequence
How does a spectrophotometer work?
1. White light passes through a monochromator which separates the light into its colored components and allows light of only a particular wavelength to strike the sample 2. The light passes through the sample and the unabsorbed portion strikes a photodetector which produces an electrical signal proportional to the intensity of light. The signal is converted to a readable output (ABS value) which is used in the analysis of the sample
Buffer used in gel electrophoresis
1X TAE - conducts electricity well (can't use water because it is not a good conductor)
What does 230nm, 260nm, and 280nm tell us?
230 nm: wavelength organic compounds are absorbed best at 260 nm: wavelength DNA is absorbed best at 280 nm: wavelength protein is absorbed best at
Why do we look at 260/280 and 260/230 ratios?
260/280 ratio: assess contamination of protein in sample (1.80 is perfect) 260/230 ratio: assesses contamination of organic compounds in sample (2.0 is perfect)
What occurs at each temperature in PCR reactions?
95°C: most cells in the sample are killed and the double stranded DNA molecules denature into single strands. 60°C: primers attach to the single strands of template DNA and outline the desired genes of amplification between them. (Primers: RNA sequences that attach to single-stranded DNA in front of and behind the desired sequence to outline it for replication (annealing of primers to DNA)) 72°C: the DNA polymerase attaches to the single strand of DNA and elongates it by adding new nucleotides, synthesizing a new complementary strand of DNA through DNA replication.
When IPTG is present...
it allows RNA polymerase to move through the lac operator and transcribe the GFP gene we placed in the MCS.
Thermocycler
machine that raises and lowers temp of DNA in precise intervals for PCR
Why is detergent in the lysis and extraction buffer?
Detergent is in this buffer to lyse the cells by breaking open the bacterial cell membrane so that the DNA is released into the solution
How does PCR work?
Done using a thermocycler to amplify DNA by cycling through a series of temperatures (makes many copies of a selected piece of DNA) REQUIRES: DNA template + DNA primers + polymerase + dNTPs + buffer (has salts and ions that keep the polymerase functioning)
Some uses of DNA cloning
Drug/vaccine development, making produce more advantageous (Flavr Savr tomatoes), gene therapy, transgenic animals for research, biopharmaceuticals 1. A real-world use of recombinant DNA cloning is the creation of blood clotting factors to help patients with hemophilia. 2. Another real-world use of recombinant DNA cloning is the production of vaccines such as the hepatitis B vaccine. 3. Creating genetically modified produce such as Flavr Savr tomatoes which were created to be able to have tomatoes that can become ripe while still on their vine.
Why do molecular biologists use plasmids?
Enables mass production of DNA
What does transformation accomplish?
Enables plasmid to enter bacteria cell
What does gel electrophoresis accomplish?
Enables you to determine the lengths of DNA fragments by comparing samples to the ladder DNA on the gel
Bacterial plasmid DNA
A circular strand of DNA other than the bacterial chromosome that contains nonessential gene that enhance survival
Parts of a spectrophotometer
A light source, a monochromator (entrance slit, dispersion device, and exit slit), cuvette, and detector
DNA gel electrophoresis
A technique used to separate DNA fragments by size due to the negative charge of the phosphates in DNA being repelled by the cathode near the wells of the rig and being attracted to the cathode on the opposite end of the rig.
Transgenic organisms
An organism whose genome has been altered by the introduction of one or more foreign DNA sequences from another species by artificial means
Limitation of Sanger sequencing
Cannot analyze large DNA samples and takes a long time
What does enzyme digestion accomplish?
Cuts DNA leaving complementary sticky ends on the insert and vector
What enzyme is involved in a PCR reaction?
DNA polymerase - carries out the process of replication by synthesizing DNA strands using dNTPs
Recombinant DNA
DNA that has been formed artificially by combining constituents from different organisms
How does IPTG induce expression of proteins?
IPTG acts as an inducer of the lac operon (like allolactose) that binds to the repressor to inactivate it so that transcription can take place on the structural genes of the lac operon.
How do we know if our transformation reaction was sucessful?
If the bacteria can grow on a plate with Kan antibiotic, it MUST have gotten the plasmid since the plasmid has the Kan resistance gene
What is the purpose of including a DNA ladder on an agarose gel?
It allows us to estimate sizes of an unknown band based on the known sizes of DNA ladder
What does a PCR reaction accomplish?
It amplifies a region of the DNA sample -- after 30 cycles of the thermocycler going through these temperatures, 1 million copies of the single stranded desired gene have been made
Why do we need 45 minutes incubation period before plating the samples?
It takes time for repressor protein and Kan gene to be transcribed and translated (expressed)
What would have happened if we did not linearize the vector DNA with a restriction enzyme?
It would not run at its correct size or at all... it would create a smear in the gel
What biotechniques are used and in what order?
LAB 7 1. Isolate Plasmid DNA vector form bacteria 2. Quantify vector DNA with micro spectrophotometer 3. Isolate insert DNA by PCR LAB 8 4.5. Linearize vector with restriction digestion 4. Gel electrophoresis of insert and vector LAB 9 5. Cleanup insert DNA using kit 6. Quantify insert DNA with PCR 7. Restriction double digest of insert and vector DNA (Nco1 & Not1 enzymes) 8. Cleanup DIGESTED vector and insert using kit 9. Quantify with PCR DIGESTED insert and vector LAB 10 10. Ligate our insert and vector together 11. Transform supplied plasmid (5ng pBIT vector in +DNA tubes) into bacteria (positive controls) 12. Transform our ligated plasmid into bacteria
What is the purpose of double digesting (using 2 restriction enzymes)?
Lessens the chance of the vector recirculizing without the insert AND enables us to control the direction of the insert being inserted into the vector
What enzyme is involved in a ligation reaction?
Ligase: seals phosphodiester bond between insert and vector to connect them due to their complementary sticky ends
Dilutions
M1V1=M2V2
Restriction sites of plasmid
Multiple cloning sites
Next-generation sequencing
Newer version of sequencing that is faster and more cost-efficient
Types of pipettes & their volumes
P10: 0.5-10 microliters P20: 2-20 microliters p200: 20-200 microliters p1000: 100-1000 microliters Serological
In this lab, we cleaned up our PCR reaction and our two restriction digest reactions. For each of these methods, list two possible reasons (like components that could have interfered with future reactions) we cleaned them up.
PCR Reaction: 1. Removing the buffer components from the insert DNA that were added during Lab 7 2. Removing the enzymes from the insert DNA that come from the bead at the bottom of the PCR tube from Lab 7 Restriction digestion of vector: 1. Removing Nco1 enzyme & Not1 enzymes from the vector DNA 2. Removing 10x NEBuffer 3.1 Restriction digestion of insert: 1. Removing Nco1 enzyme & Not1 enzymes from the insert DNA 2. Removing 10x NEBuffer 3.1
What controls should be used in transformation?
POSITIVE: DNA+ Kan, IPTG & DNA+ Kan · If these do not work, there is a problem with the experimental design NEGATIVE: DNA - Kan, IPTG, & DNA- Kan · Ensures everything except IV is functioning properly
How to correctly load and read a cuvette in the machine
Place the clear face of the cuvette containing the blank solution facing the front of the machine, then press autozero, then place the first cuvette with the solution the same way as the blank; repeat this process, zeroing in between trials
What does the nucleic acid clean up kit accomplish?
Removes reagents that would affect downstream reactions
What other methods could be used to help confirm that we cloned the correct gene?
Restriction digest and gel electrophoresis, blue-white screening (color of colonies - blue colonies do not have insert, white colonies do), PCR
What restriction enzymes are involved? Where do they come from? How do they work?
Restriction enzyme: DNA cutting enzyme that cuts DNA at specific recognitions sites Came from bacteria as they were originally used as a defense mechanism against bacteriophages inserting their foreign DNA int bacteria cells Used in our experiment: Not1 and Nco1 enzymes
What does ABS represent?
The ABS values indicate how much of the light the sample absorbed and did not allow to pass through to the detector. A higher ABS value corresponds with a higher concentration, and a lower ABS value corresponds with a lower concentration.
What does the silica membrane do (silica membrane in spin column)?
The DNA binds to the silica membrane via a SALT BRIDGE, enabling us to isolate the DNA as the flow through is discarded down the sink (to remove the DNA from the spin column, we add an elution buffer and centrifuge and then the DNA is in the flow through)
What role does the antibiotic play in being in the media plate?
The antiobiotic Kan enables us to determine if you bacteria have the plasmid within them as this plasmid has a Kan resistance gene so the bacteria will only survive if they successfully received the plasmid.
Why should you not vortex in plasmid isolation?
To not contaminate plasmid DNA with genomic DNA
Why did we heat shock the bacteria?
To stress the cells out so that pores open in their cell walls, enabling the plasmids to enter
How does transformation work?
Transformation: the process by which bacteria takes up foreign DNA from its environment by being heat shocked Heat shock: put the tubes in a water bath at 42°C for 30 seconds - cause pores in the cell wall that enabled the plasmid to enter the bacteria cells
When performing a transformation reaction, why should you use a Ca+ solution?
Use a Ca+ solution as Ca+ neutralize the negative charges of the plasmid and bacterial cell wall, dissipating electrostatic repulsion and weakening the cell wall
How do you read DNA gels?
Use loading dye so you can visualize the fragments under UV light and compare how far they travelled to the ladder DNA to determine size
Vector vs insert (and the ones we used in lab + their size)
Vector: plasmid DNA used as a tool to make more species of or produce protein from a certain gene (PEt-41a - 5,933 BP) Insert: the DNA fragment you are going to clone (eGFP - 734 BP)
Metric system units
Volume: liter (l) Mass: gram (g) Length: meter (m) Temperature: Celcius (°C)
Why is the wavelength important?
You need a spectrophotometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths
What does a ligation reaction accomplish?
ensures an intact sugar-phosphate backbone between insert and vector
Why do you incubate plates upsidedown?
to prevent contamination from condensation
Metric conversion
·Kilo (k): 103 ·Hecto (h): 102 ·Deca (da): 101 ·UNIT ·Deci (d): 10-1 ·Centi (c): 10-2 ·Milli (m): 10-3 ·Micro (u): 10-6 ·Pico (p): 10-9
