Again You Are Making Ampicillin Plates Using Lb Agar

DNA TRANSFORMATION OF Leaner-AMPICILLIN

Prepared by the Role of Biotechnology, Iowa Country University

Revised viii/2000

Instructor Training
Sterilization Of Supplies
Preparation Of The E. coli Starter Plate
Clean Up After The Laboratory
Student Instructions

Teacher Grooming AND INSTRUCTION GUIDE

Preparation for the Dna transformation experiment should begin at to the lowest degree 24 hours in advance of the laboratory period.

The following supplies can be provided to the class in groups of three students:

2 microcentrifuge tubes (1.5 ml) containing 2 drops of sterile CaCl₂ and labeled "CaCl₂". The tubes can be put in the aforementioned water ice container used to provide the DNA to the group of three students.
1 aluminum foil packet containing 4 sterile toothpicks
iv sterile plastic pipettes from the Office of Biotechnology
1 aluminum foil package containing iv sterile paper clips that are large and smooth. The clips should be opened into a ninety^o angle and the pocket-sized end bent to close it.
1 Sharpie marking pen
ane glass test tube with a cap (provided by the Office of Biotechnology) containing 2 ml of sterile nutrient broth and labeled "Broth"
ii petri dishes containing merely nutrient agar and labeled "No Amp" on the bottom
two petri dishes containing food agar and the antibiotic ampicillin. The dishes should be labeled "Amp" on the bottom. (Petri dishes provided past the Part of Biotechnology)
two copies of the laboratory instructions, 1 for each student

The following supplies can be shared past three students:

one petri dish containing colonies of E. coli (MM294)
1 microcentrifuge tube (1.5 ml), labeled "P", containing iv drops of plasmid DNA that is placed on water ice to keep cold until used. The tube should exist labeled "Deoxyribonucleic acid".
ane container for used toothpicks

The teacher should have available for the entire class:

one incubator for the petri dishes gear up at 37^o C or less. It is hard to maintain the temperature precisely unless a research incubator is used. Prolonged temperatures above xl^o C will kill the leaner. Temperatures lower than 37^o C will result in slower growth of the bacteria, just will not kill them.
1 Sharpie marking pen
Containers for placing tubes on ice afterward Deoxyribonucleic acid has been added, such equally a styrofoam loving cup.
Containers for the 42^oC h2o bathroom, such equally a styrofoam loving cup.

STERILIZATION OF SUPPLIES

Sterilization of packets of toothpicks, and paper clips tin be achieved past wrapping each particular in aluminum foil, labeling the contents with a marking pen, and
(a) blistering them in an oven at 350^o F for 15 minutes

(b) putting them in a pressure cooker at 15 pounds for 15 minutes

(c) placing them in an autoclave for fifteen minutes.

The pressure cooker and autoclave should be at the desired pressure for the 15-minute period. After the packets have cooled, they should exist stored unopened at room temperature. The students should be instructed when opening the packets to bear on simply that part of the object that will not come up in contact with the solutions or petri dishes.
Sterilization of the 1.v ml microcentrifuge tubes can exist achieved past wrapping in aluminum foil all of them needed past the teacher to prepare the supplies for the students. The tubes can be:
(a) baked at 250^o F (they cook at 350^o F) for xxx minutes
(b) put in a pressure cooker at 15 pounds for 15 minutes

(c) placed in an autoclave for xv minutes.
Calcium chloride. Dissolve 0.75 1000 of CaCl_ii into l ml of distilled h2o in a labeled 100-ml drinking glass bottle with a cap. Continue the cap loose and place it in:
(a) boiling water for 30 minutes

(b) a pressure cooker at 15 pounds for xv minutes

(c) an autoclave for xv minutes.

Allow the bottle to cool until information technology is comfortable to concur, cap it tightly, and shop in a refrigerator until used.
Ampicillin solution. For each 1,000 ml of Amp agar to be prepared, dissolve 50 mg of ampicillin (sodium salt) in 1 ml of cool sterile distilled water. The h2o tin exist sterilized past placing it in a glass bottle that is not more than half full, putting the cap on loosely, and using i of the procedures described for the calcium chloride. The sterile water should exist stored in the refrigerator until information technology is used to brand the ampicillin solution. The ampicillin solution should not exist prepared and stored in advance for an extended period. The solution should be prepared and put in the refrigerator immediately earlier the nutrient broth solution (Item 6) and the agar plate solution (Detail vii) are prepared.
Plasmid DNA solution. The plasmid DNA used in the laboratory has a gene for ampicillin resistance. The plasma Dna is obtained from the supplier in a concentrated solution, which has to be diluted to 0.005 ug/ul for the DNA transformation experiment. The Dna should exist distributed to the students in tubes kept on ice. Whatever unused 0.005 µg/µl Deoxyribonucleic acid can be stored in the freezer for future use. In a self-defrosting freezer, the DNA should be put on ice in an insulated container, such as a Thermos jar.
Nutrient broth solution. Calculate the amount of nutrient broth that is to supplied to the students and add actress for spillage and other factors. Weigh 25 mg of LB premix /ml of distilled water into a bottle and label it. Add the appropriate volume of distilled water to the bottle. The canteen should not be more than than half total so that it does not boil over during sterilization. With the cap of the bottle loose, use one of the sterilization procedures described for the calcium chloride (Item three). Subsequently the LB has cooled and is comfortable to hold, cap it tight and store in a refrigerator until it is dispensed to the class.
Before the class, put 2 ml of the LB into drinking glass test tubes, leave the caps loose, and identify them in an appropriate rack in boiling water for 30 minutes to sterilize them. After the thirty minute-flow, remove the tube rack from the boiling water, allow the tubes cool, so tighten the cap. Unused broth can be reboiled and stored in the fridge for future apply.
Agar plates. Two types of agar plates should prepared: Without ampicillin " No Amp", and with ampicillin "Amp". Prepare separate solutions for the "No Amp" and the "Amp" plates. For each type of plate, 25 ml of agar solution will exist required per plate. Label the plates on the underside, not the lid, before they are poured.
"No Amp" plates: Prepare 3 "No Amp" plates for each group of three students; 1 for preparation of the starter culture and 2 for each pair of students to utilise for transformation. Information technology is best to gear up well-nigh 5 extra plates for the entire class in example contamination occurs in one or more of them. Place the required volume of distilled h2o in one or more glass bottles with caps. The bottles should not exist more than half full. Add together 25 mg of LB premix and fifteen mg of agar /ml of distilled water. With the caps loose, sterilize the solution by one of the methods described for the calcium chloride (Particular 3). After sterilization, the bottles should be swirled to mix the solution and cooled at room temperature to 55^o C, which is when the bottles tin can exist held without an insulated glove. The petri dishes labeled "No Amp" should be poured immediately. The bottom of the dish should be covered with the agar. Agar begins to solidify at about 45^o C, therefore, it is important to pour the plates as chop-chop as possible. If the "No Amp" agar does solidify, it tin can be reboiled and used again. Rinse the bottle with a big amount of tap water immediately later use and so that the agar does non solidify in it or in the sink.

"Amp" plates: Gear up 2 "Amp" plates for each group of 3 students. Follow the same procedure as for the "No Amp" plates until the agar has cooled to 55^o C. Add 1 ml of the ampicillin solution (Item 4) per liter (1,000 ml) of solution, swirl to mix, and cascade immediately the plates labeled "Amp". If the agar solidifies, it cannot be reheated because the ampicillin will be destroyed in a higher place lx^o C.

Permit the "No Amp" and "Amp" plates to harden for virtually 30 minutes or until the agar has a milky or opaque advent, then turn the dishes upside down (hat downwardly, agar up). If they are to be kept for more than two days, store them upside down in a refrigerator. The plates can be kept refrigerated for a month.

Notation: People differ in their sensitivity to temperature and a teacher may prefer to mensurate the temperature of the agar to determine when 55^o C is reached, especially for the solution to which ampicillin is added. It is not possible to put a thermometer into the heated agar solution because it will become contaminated. There are ii alternatives that tin can be used.

(A) The bottle of agar can be put into a container with the same volume of absurd tap h2o as the volume of the medium inside the bottle. When the temperature of the tap h2o reaches 55^o C, the contents inside the canteen should be at a similar temperature.

(B) The bottle of agar can be put into a hot h2o bath at 55^o C and allowed to stand for xxx minutes.

PREPARATION OF THE E. COLI STARTER PLATE

I petri dish containing live E. coli is needed for each group of four students. A strain of E. coli should be used that does not accept resistance to ampicillin.

Employ a sterilized transfer loop, a paper clip bent into a loop and sterilized, or a sterilized toothpick. Use the device to touch a colony of bacteria from a petri dish or test tube. Spread the bacteria on the plate in a zig-zag pattern to obtain individual colonies equally the concentration of bacteria on the transfer device becomes less. Incubate the plates at 37^o C for 24-36 hours. Colonies should abound to the size of this 0 for use in the lab procedure.

CLEAN UP After THE LABORATORY

Sterilize used toothpicks and 1.5 ml microcentrifuge tubes before placing them in the regular trash. Sterilize the pipettes earlier washing them. Sterilization can exist accomplished by placing them in boiling h2o for 30 minutes, autoclaving for 15 minutes, or putting them in a force per unit area cooker at 15 pounds for xv minutes.

Wash glass bottles, pipettes, and paper clips for future utilize.

Petri dishes tin can exist burned, if convenient. If non, freeze the plates overnight or allow them to dry out out in the fridge for 1 month, then wrap them securely in a plastic purse and place them in the regular trash.

Educatee INSTRUCTIONS

Genes control the traits that living organisms possess. Bacteria, such as E. coli, have genes on their chromosome and on a small circular piece of Dna chosen a plasmid. Genes can be transfered from one bacteria to another on the plasmid by a process known as transformation. In this experiment, a plasmid with a gene (Deoxyribonucleic acid) for resistance to the antibiotic ampicillin will be used to transfer the resistance gene into a susceptible strain of the bacteria. The same technique is used to transfer genes (Dna) for production of insulin, growth hormones, and other proteins into leaner. The transformed leaner are used in fermentation to produce commercial quantities of the poly peptide for treating diabetes, dwarfism, or other uses.

You will piece of work with two other people in conducting this laboratory.

PreLab DAY 1

Step ane. Apply a divide sterile toothpick to transfer a colony of Eastward. coli about the size of this 0 into each of two tubes of calcium chloride. Use the toothpick to stir the cells vigorously and thoroughly into the solution. The solution should announced milky. Shut the caps of both tubes and discard the toothpicks into the container provided for that purpose. I person in the pair should label one of the tubes "B1". The other person should label the other tube "B2".

Step two. Identify the tubes back in the ice and place the container of ice with tubes back in the refrigerator. (DO NOT FREEZE.) (The common cold calcium chloride, in the tubes, conditions the surface of the bacteria for DNA uptake the following day.)

DAY 2

Footstep 1. Finger flick tube to resuspend cells.

Step ii. Open up the tube labeled "B1" and with a sterile pipette add one drop of solution from the "P" tube. Close the tube. Exercise not add anything to the tube labeled "B2". (The plasmid DNA, from the "P"tube, added to the tube has a gene for resistance to ampicillin.)

Pace three. Place the tubes on ice for 15 minutes. (The cells are kept cold to prevent them from growing while the plasmids are existence absorbed.)

Step 4. Remove the tubes from the ice and immediately hold them in a 42^oC water bath for 90 seconds. (The marked temperature change causes the cells to readily blot the plasmid Dna).

Step 5. Utilize a sterile pipette to add 5 drops of sterile food goop to each of the tubes. Shut the tubes. Mix past tipping the tube and inverting it gently (The leaner are provided nutrients to help them recover from the calcium chloride and heatshock treatments).

Note: For better results allow prison cell recovery at 37^o C for whatever corporeality of actress fourth dimension, 20 minutes preferred.

Stride six. Label the underside of the four petri dishes with your name. On ane "Amp" plate, print "B1" and on the other "Amp" plate print "B2". On i "No Amp" plate print "B1" and on the other "No Amp" plate print "B2".

Pace 7. Use a fresh sterile pipette to identify 3 drops of cell suspension from the tube labeled "B1" onto the middle of a petri dish labeled "Amp"/"B1" and 3 drops to the center of a dish labeled "No Amp"/"DNA". Apply another fresh sterile pipette to identify three drops of prison cell suspension from the tube labeled "B2" onto the center of the dish labeled "Amp"/"B2" and iii drops to the center of the dish labeled "No Amp"/"B2". Apply a fresh sterile paper clip to spread the liquid evenly across the surface of each plate. Do non touch the part of the newspaper clip that comes in contact with the agar.

Step 8. Incubate the plates upside down for 24 hours at 37^o C.

Step 9. Clarify the results of the transformation by placing the 2 plates labeled "Amp" and the 2 plates labeled "No Amp" together. (The plate labeled "Amp"/"B2" should not take bacterial growth because the bacteria are killed because they did non accept resistance to the antibiotic ampicillin. Bacterial growth on the "Amp"/"B1" plate is from cells that took upward plasmids added in footstep 2 and that became resistant to ampicillin. There is extensive bacterial growth on both of the "No Amp" plates because the antibiotic was non present and both resistant and nonresistant bacteria could grow.)

Prepared by the Office of Biotechnology, Iowa State University revised 8/00

Again You Are Making Ampicillin Plates Using Lb Agar

Source: https://www.biotech.iastate.edu/publications/lab_protocols/DNA_Transformation_Amp.html

Stoddard Porang