Lab Introduction, Safety and Group Assignments

Here are concise study notes based on the provided text:

• Choose a seat at a bench, where you sit determines your lab partner.
• Wipe down your bench with Quat and wash your hands.
• This section covers Assigning groups, preparing LB media, class intro, safety/rules, the syllabus, the lux operon, lab calculations and finishing making media.
• Contact Stephen Lumsdaine with any questions
• The appropriate email address is [email protected]
• Stephen Lumsdaine can be reached via email only, not Canvas
• Responds within 48 business hours (not weekends)
• Office hours are by appointment, so email questions/topics in advance.

Other People You Need to know

• Garrett Huff is there for Autoclaving, Lab Maintenance, & Media Production.
• Jessica Pyle is a lecturer.
• Haley Dylewski is a lecturer

Group Assignments

• This lecture will cover group assignments for this section

Lab Etiquette

• Upon entering the lab, place belongings (except lab manual, writing utensils, calculator, and writing paper) in a cabinet, cell phones are prohibited.
• Wipe benches with Quat cleaner.
• When organisms or reagents are out, put on a lab coat, tie back hair (no hats), and grab a lab mask, keep the lab mask in your pocket for later use.

LB Preparation

• Crucial to the growth of pure cultures of E. coli
• Prepare 200 mL in a 500 mL beaker
• Use LB Broth, not LB Agar, otherwise it will solidify
• After dissolving chemicals, create 10 aliquots of 10 mL
  • Do not add ampicillin until the day of inoculation
  • Same weigh boat can be used if everything that touches it is going to be mixed
• Agar plates should be made with the remaining 100 mL
  • Use "Agar,” not agarose
  • Add ampicillin after autoclaving when the medium has cooled
• Add foil and autoclave tape; bring up to autoclave

Making LB Broth

• To mix H2O and LB powder for LB Broth, use a total volume of 200 mL and calculate the mass of LB powder based on 25 g powder/1 L water
• For 200mL of LB Broth powder the calculation is 0.2 L * (25 g/ 1 L) = 5 g LB powder

Todays Activities

• Today you will make 10x 10mL tubes of LB broth plates
• Mix H₂O, LB powder (add 5 g of LB powder) in 200 mL total volume.
• Split half into ten tubes and other half into Erlenmeyer (add 1.5 g agar to Erlenmeyer, 1.5% = 1.5 g Agar)
• Also autoclave one 10mL Tube of water
• After this, autoclave and allow media to cool before you add Ampicillin to Erlenmeyer Flask, finally Pour Plates and store broth

Course Matericals

• There is no textbook for this class.
• Canvas modules: Background information, Manuscripts, Protocols/Reagent Information
• A bound notebook is required for lab work, it must be permanently bound (no spirals)
• Read/understand the material before coming to class.

Goal of Lab

• You will learn how to do independent research
• Students will learn to adhere to protocols, troubleshoot, and work within a team.
• The semester project is to clone the lux operon from alivibrio fischeri into Escherichia coli.

Molecular Cloning Project

• Clone the lux operon from Vibrio fischeri into Escherichia coli is the Task
• This projects goals are to Learn how to perform and apply various cloning techniques, and to make sure you perform each step yourself
• You can work Independently within your team but I will teach you what I can up through the Fall break and am here for guidance.

Open Lab Hours

• Due to the Independent nature of this course you will need to come outside of class time.
• Open Lab hours: Monday: 7am-3pm, Tuesday & Thursday: 7am -11am and Wednesday & Friday: 7am – 11am
• There are classes in here on these days so Do not work during another class period or interrupt another class
• You cannot work in the lab in the evenings or on weekends!!!
• The last daily call for autoclaves is at 12:30p.m. daily. this cycle takes 1.5 hours so Autoclave at the beginning of class.

Rules and Safety

• In this lab you will need to follow all the stated rules or face penalties to your grade
• For your own safety you will need to wear: closed-toed shoes, long hair must be tied up, a lab coat and gloves, a mask, make sure there is no food!
• Treat all equipment with respect
• The rule of thumb is to "Clean bench area upon arrival & before leaving, (Spray Quat 50, paper towels ect.) Disinfect hands before and after any micro work
• Contact lenses are dangerous!
• And always protect youself and remember, "This lab must look the same when we leave as it did when we arrived".
• Waste Handling is also critical, in this lab you need to correctly dispose of all of your waste materials
• Put Biohazard waste in red bins, NEVER include glass into the red bins
• Make sure gloves go specifically into biohazard bags, NOT the trash can
• Serological pipettes must go in to the small Quat bins and Never thow the Ethidium Bromide w/ the general biohazard waste

Grades

• Grades: Lab work Prep is worth 25 pts, and performance is worth 35
• 60pts of 720pts total and is determined by: reading protocols, attending class, helping others, cleaning up, being respectful

Grades: and Syllabus

• This Classes attendance is worth 10 pts, and being late will subtract 5
• Excused Absenses must include Jury duty, death in the family, or UT sanctions
• To obtain an excused absense: You must have proper documentation to contact Instructor within/before class

Grades Content

• In the Lab notebook it is important to randomly record 40 pts of the content Randomized look at Table of contence 4x each.
• You must have a short intro to understand what is what. "So can I repeat and obtain, what did I proceed?"
• Record the Data, Images and what graphs happened.

Lab Notebook specifics

• Record all your data with PEN (only)
• Write the date next to each entry
• Each title must be descriptive, make step by step instructions like making cheese

Syllabus cont.

• When recording results and conclusions remember to tape images not staple
• Conclusions are the single most important aspect. "What would you do different than previous"

Grades Mastery and Grading

• 100 pts for exam 1 Midterm total. Content mastery is a big factor "is it understandable"
• In content you must ask: "Why are you doing thses techniques" and "Do you know the biology well"
• The format is short and sweet with a good amount of understanding.

Content part 2

• The quiz is there the PREPARE you for mastery and is worth 40 pts
• Unannounced at any time. "Pre and post lab readings?"

Final

• Exam 2 for 100 pt total has opened books and is designed "can you apply knowledge"
• These will have open ended structures for the best understanding.

Grades: literacy

• Grade determined by 45PT total scientific literacy
• The critical summary must include 3 assingments with descriptions of actions related to the previous figure.

topic summary

• Chose a current manuscript at the beginning with no reviews. Have copies to submit due on canvas for 75 total points.

grades grading

• Grading is performed by peer review and is worth 200 total.

grading

• Final Lab Report: May 7 at 5:00p.m. (0% if turned in late) Each must be turned in as a hard copy in class as well as submitted to Canvas Turn it in plagiarism checker

plagarism

• you must have a zero tolerance policy when you dont give credit during plagarism.

grading

• all ai content in this class is considered plagiarism and will cost you any points at all.

grading others

Consult the other protocols FIRST, then receive 0 credit. read all you can first to reduce questions.

making stuff

preparation of tranformation media

Making stuff today

Mix H₂O and LB powder and Split half into ten tubes and half into an Erlenmeyer. after that mix everything up.

Making stuff next class

the Friday after we autoclave and pour.

Measurments

L = Liter measurment using 10-³ for ML and 10-6 for UL... these measurments are the basics of chemistry and most labs.

Volume and %

• Dry reagents can be expressed as a % solution like 50 ml. (step 1 and 2 % to decimal"

Adding Agar

• Lets say you want to make 500mL of 2% agar...
• 2% as a decimal = 0.02" and final should be be mililiters

lab prep

LB medium = Lysogeny Broth = a liquid, and LB agar has 1.5% Agar powder.

Fungal growth

• Protocol for YPD media and is also used for fungal growth following a extract.
• Make 150mL of this media and "add the needed components?"

math

• Adding ampicillin or other drug markers Stock solutions are at higher concentration than working solutions such as c1v1 = c2v2

Lab info

• Remember to write your names or initials it on your freezer box, the other classes also have a group 1/ 2/ 3 etc.

info making

• for all LB agar plates: Stock Ampicillin (50 mg/ml) to dilute final and USE C₁V₁ = C2V2 all the day. make sure that you keep your name on it at all times.

Making the media

Make Good Vibrio Medium (GVM) plates and broth with adjusted solutions to volume.

making media

“ 1 mL of water = 1gram", or have the chance to test and do it over.

rules about lab

after you use the correct chemicals make sure you perform the right procedures, to not contaminate it.

Class guide

“Bring a sharpie and notebook" and understand them! read the protocol.

Intro of other notes

All of the notes are taken into account for the rest of the session and are very important.

overview

• the LUX code determines how things will work and how each vector acts alone the side and what process with the bacteria they do.

Lux notes

Alivibrio fischer heterotrophic bacterium and Bioluminescent symbiont of Hawaiian Bobtail Squid. the lux is from its gene.

lux operons

• The lux codes emits light and requires certain genes to regulate it.

Sensing

• Quroum sensing, or self regulation, are controlled through multiple means.

Knowing

• Luxl = Enzyme that produces AHL, AHL = Binds to LuxR and allows it to bind to lux box, LUX controls the positive polymerase.

The rest.

• 490mm is light for bacteria and in all schemes there was some form of light control
• Quorom sensing need to be high at 10^9 -10^10 cell per ml
• Also 10*4 a unit of quanta per second.

last of notes.

• 8/30/23 notes and all notes before that are just a reference point
• Make some sterile water today if you did not last lab.

more notes

• you have two extract gDNA and you need to follow it. read the book with the code thing.

Math lecture.

• Lets say you have a 500ml and YPD and the following is 250
• so take that 5/2 is the extract that you need.

figure reference

There does need to be data of some form presented not just a random graph.

open lab

The last time to do any lab activity is near open hours before it shuts down.

General notes

Remember to set up inoculations of V. fisheri for class Friday in order to set bacteria growth for plasmids, you will see this every step of the way.

basic

A piece of DNA can transfer its information into a bacterial cell .

vectors

Vectors are our friends and plasmids allow for the construction of things, and help to provide a host environment.

Plasmids.

Origination is the most common trait to keep plasmids working. Multiple cloning is good in newer sets and high tolerance is usually an important factor.

plasmid 2

"High: Typically used for increased protein expression" versus Low: Usually used when concerned about production of proteins that may kill the cell" is also important to maintain functionality.

tags

Expression Vectors provide epitope and easy to get tags.

More tags

More codes like poly hIS tag and bOTAN are added to many aspects of bacteria/yeast genes.

markers

A.R and AU are the 2 forms of marker used in these settings.

Runaway Vector

Replicate just as expression constructs do up to 34 °C but Uncontrolled replication of plasmids when ≥39 °C for disruption.

Large.

Large Plasmids (>15 kb) is way to hard to use due to cell lysis, versus smaller means stronger options.

notes for plasmid..

Carefully review data, do not vortex warnings, longer centrifuges and pre warmed material.

and finally the drop

Remember Three numbers concentration purity contmaination

Genes and knowing

Luxl = Enzyme that produces AHL,AHL = Binds to LuxR and allows it to bind to lux box in general.

general thoughts

These genes have an affect on DNA regulation in code areas.

final words

These genes are known for "releasing" some form of light on a certain level.

general extract process

Cell lysis -Protiens precits - precipi taiton

DNA prep

Phenol:Chloroform:Isoamyl Alcohol (25:24:1) and use the solution with DNA or RNA.

precip

salt water has great power and is able to create a hydrogen bond with H2.)

Polartity

EtOH polarity < H₂O polarity is the start of DNA precip to get the right bonds going.

Extract all you can

• Alkaline Phosphatase: removes 5'-PO from the DNA terminus
• DNase I: hydrolyzes phosphodiester bonds
• B-Agarase I: digests agarose

storing extracted matieral

Storing DNA is best as sterile until you extract it with 10 MM 4.5

recap

Cell lysis helps get our DNA out

• Protiens must be cleared
• precit
• Dna also helps
• Remember to mark and use.

lab guide

"You take from the frezzer and then prep as best you can."

Project time

After these two processes we extract and get to test it on 4.1

notes from lecture

V. fischeri also help do everything or even read the modules again from canvas.

vectors and guide

Each plasmid contains a "luz: for example.

plasmids and test

These restriction tests " cut DNA code" is known but it does what's on it.

Restriction guide

After all that said... EDTA and co factor are amazing for this task.

cut then do

The product at the end can be either very clean or with random lines.

cutting code

A- the first "is often our vector cut out

• this does what we call in basic sense with the process"

Saftey!

Is very important to look at the "Ethium Bromide" and is has dangerous effect on humans!.

Ethium is NOT. a tool to be played with

other tips during making

When you make it dont over stress it. Make the right buffer but do not over run the cord

after all the procedures..

You test again to see which type of DNA to use for testing codes. and if its good

Structure tests

The linearized is a good test to maintain its accuracy and will tell the success rates.

What is and what isn't

Be very aware in these processes that gDNA to get the code on ether ends. And sall, it does what it can in testing pGEM.

Other important notes to take

• alkaline Phosphatase: removes 5'-PO43- is very important.
• B-Agarase I

After

When all said in done " Short- because it is not super long.

finally!

Always rember to ""Clean the ethium"" """ And if we did it then we add and test by running a chart

all set good

• A high high cord with 3d40k Make sure to note all the following
• Don't over or under And remember the chart well

transformation

• Bacteria take up all things

all things

What and which is it.

What

We and the

final

All points needed and the right test for coding the whole time.

transformation

The code to select is at 100

Transformation 223

That we go out to this point with another factor known at the same time. Remember, we are on something.

• Then again, do or do

Code and 213

The transformation With the codes that give the main function in the short long

Next steps.

This is going to be fun, we will all do it well and move forward thanks.

next steps

Again the code is very important Remember do not mix this together.

coding tips

"A little mix here and there and now we have the code" With that code and other tips. We do the main function we must all do. "

Important tips for now..

"These things and data are on the board," You will also follow it correctly. Enjoy the test

Main notes and all that jazz

To calculate transformation you must look over all your data for this step. and then it does need it all Transformation efficiency is only calculated correctly All that will help and to the end