BS7101: Practical Molecular Biology And Microbiology - Introduction to Cloning - Lab Report Writing Assessment Answer

Subject Code: BS7101 Internal Code: 1AIEIB

Lab Report Writing Assessment Answer

Assignment Task: BS7101 Subject Introduction to Cloning Introduction This practice will be the first of a series of three practicals within the BS7101 module where the students will learn and practice how to successfully clone a vector using competent bacterial cells, how to purify the vector and perform a restriction digest and separate the fragments on an agarose gel. Content In this practical you will perform:

1. Preparation of competent cells
2. CFU determination

1. Preparation of competent cells Intro In this section the student will treat E.coli bacteria (DH5alpha) to produce cells competent for transformation, to be used all throughout the 3 practicals. The term ‘competent cells’ refers to bacteria that have been treated so that plasmids can be inserted into them. Effectively their cell membrane has been made temporarily more permeable. You are provided in the ice bucket with Eppendorf tubes containing bacterial cultures that have been cultured at 37°C until they reached log phase growth and then transferred to ice. Keep the bacteria as cold as possible through the whole procedure. Protocol

1. Centrifuge two eppendorfs with bacteria at 5,000g for 2 min in a refrigerated centrifuge.
2. Discard the supernatant using a pipette while avoiding disturbing the pellet.
3. Resuspend the bacteria with 800 ul of ice-cold 50 mM CaCl2, incubate on ice for 15 min.
4. Centrifuge again at 5,000g for 2 min. and store at 4°C.

The cells would now be ready to receive the plasmid.

2. Determination of CFU (colony-forming units)

Intro It is often necessary to estimate the concentration of bacteria in liquid culture. This can be done in two ways: 1) a crude way is to look at cell density in terms of light absorbance, 2) another way is to calculate the colony forming units (CFU) Protocol

1. Prepare serial dilutions of the bacterial culture provided. Each dilution will be 1 mL of volume. Prepare 5 Eppendorf tubes labelling them as follows:
   • 1 (stock bacterial culture provided)
   • 2 (1:10)
   • 3 (1:100)
   • 4 (1:1000)
   • 5 (1:10000)
2. Use the knowledge acquired during the Introductory practical in order to prepare the serial dilutions.
3. Label 5 agar plates to correspond to the serial dilutions.
4. Spread 100 uL from each dilution on an agar plate (correctly labelled) with the sterile hockey stick.
5. Incubate overnight at 37°C (after the colonies have grown, the plates will be stored at 4°C till the next practical).
6. Use the remaining volume of the serial dilution to measure the absorbance at OD600 by spectrophotometry. Save the results.

Determining cfu (this part will be performed in the following week, practical 6) At the practical in the following week the plates will be used to determine bacterial growth:

1. Observe all the plates and choose the one with 30 to 200 isolated colonies.
2. Multiply the number of colonies on the plate by 10 to calculate the number of cells per mL of culture from the dilution tube used.
3. Multiply the number from Step 2 by 10^(plate number) to calculate the number of cells per mL of original culture. The value of 10^(plate number) is the dilution factor of the culture used to inoculate that plate.
4. Compare the cfu calculated in step 3 with the OD600 values that you measured for each of the serial dilutions.
5. Produce a standard curve with the cfu calculated and the OD600 measured.
6. This standard curve can be used to estimate the cfu/ml from OD600 measurements and should look similar to the curve below.

The video below contains interesting explanations pertinent to the estimation of the cfu. Week 6 Subject Transformation of competent cells with pBR325-COX2 plasmid Introduction This practical follows the one done in week 5 where competent cells where prepared. In this practical, competent cells will be “transformed” with a vector carrying the COX-2 cDNA, meaning that you will be able to introduce the vector into the bacterial cells by a specific procedure. After that, the transformed cells will be spread on an agar plate containing specific antibiotic, where each bacterium will produce one colony that will be used to obtain a new “clonal” culture of transformed bacteria containing the vector. Before starting, you will observe the serial dilution you prepared last week. Refer to the section “2.Determination of CFU (colony-forming units)” to calculate the CFU. Content In this practical you will perform:

1. Transformation of competent cells with the pBR325-COX2 vector
2. Bacteria plating on agar plates
3. Colony pick.
4. Inoculating a culture for a miniprep.

1.Transformation of competent cells with the pBR325-COX2 vector Intro In this section, you will transform the competent cells you prepared in the last practical with a plasmid carrying the ORF of the gene COX-2 (pBR325-COX2), a gene involved in a number of inflammatory events. To insert the plasmid into the cells you will employ the technique of “heat shock”, which will temporarily create holes in the bacterial surface allowing the plasmids to get in. The plasmid will carry a “resistance gene”, meaning a gene able to confer to the resistance of the cells to a specific antibiotic so that only the successfully transformed cells will be able to grow in the presence of that antibiotic. In this case, pBR325-COX2 will carry the ampicillin resistance gene. The competent cells will be provided to you in an ice bucket. Keep the cells in the ice throughout the whole procedure when not stated differently. A few microliters of plasmid will be provided too. In this section, each pair will work with 2 samples of competent cells (transformed or not transformed). Protocol

1. Keep the cells on ice until thawed.
2. Add 1 to 5 ul of plasmid to one of the tubes containing the cells (the exact amount will be specified during the practical, depending on the plasmid concentration). Label the tube accordingly.
3. Do not add plasmid to the second tube, but process it along the tube containing the plasmid (no-plasmid control)
4. Leave the samples on ice for 30 min so that the plasmid can cover the bacterial surface. In the meantime, set a water bath to 42°C.
5. Set a stopwatch to 30 s. Place the samples in the water bath for exactly 30 s (Heat shock), then bring back on the ice.
6. Incubate the cells on ice for 3 min.
7. Add 0.9 mL of fresh LB without antibiotic and incubate at 37°C for 45 minutes to allow the growth to start.
8. Now your competent cells should have been transformed with your plasmid of interest.

2. Bacteria plating on agar plates Intro After the transformation, you will plate the cells on agar plates containing the specific antibiotic which the plasmid conferred resistance to (Ampicillin in the case on pBR325-COX2). You will plate two different amounts of transformed cells to make sure that you will not obtain confluent colonies of too few colonies.   Each bacterium will produce a single colony that you will use to create a new clonal culture of cells containing the plasmid. After the growth, the agar plates can be stored at 4°C for a few month for future use (to pick other colonies if needed). Protocol Two agar plates with Ampicillin per sample will be provided. Let them warm to room temperature before plating.

1. Label the plates accordingly with the type of sample and the amount of cells given (for example 1/10).
2. Add 100 uL of transformed cells to one plate and spread the cells using a plastic spreader.
3. Centrifuge the remaining transformed cells at 5000g for 10 minutes.
4. Gently pour the supernatant out. Flick the bottom of the tube to resuspend the pelleted cells.
5. Add the whole volume (around 100 uL) to the second plate labelled accordingly with the type of sample and the amount of bacteria (9/10 for example). Spread the cells using a plastic spreader.
6. Spread the non-transformed control cells onto a third plate (labelled accordingly), to check that no colonies will grow.
7. Close the plates and put upside down at 37degrees overnight.

On the following day the plates will be placed in the fridge at 4°C until the week 7 practical. 3. Colony pick and culture for miniprep Intro Now that colonies have grown, compare the plates with the transformed bacteria with the plates containing non-transformed bacteria. The second plates won’t likely show any colony as the bacteria did not acquire resistance to the antibiotic. This Report Writing Assessment Answer has been solved by our Report Writing experts at My Uni Paper. Our Assignment Writing Experts are efficient to provide a fresh solution to this question. We are serving more than 10000+ Students in Australia, UK & US by helping them to score HD in their academics. Our Experts are well trained to follow all marking rubrics & referencing style. Now observe the plates with the two different dilutions of plated cells (1/10 and 9/10). Choose the one where you can see a considerable amount of colonies spread enough to pick a single colony without the risk to pick a close one. From that colony you will establish a new clonal bacterial culture with the plasmid of interest, which can be used to extract an amount of plasmid sufficient for later applications (for example restriction analysis, cloning of new genes, transformation of mammalian cells). Depending on the amount of culture (i.e. of liquid) you will set, you can produce miniprep (up to 3 ml), midiprep (up to 25 ml), maxiprep (up to 100 ml). The different volumes will yield different amounts of plasmid according to specific downstream applications. Protocol You are provided with agar plates with grown cells.

1. Prepare one tube with 3 mL of fresh LB medium containing antibiotic, and label it accordingly.
2. Choose a colony to pick that is far from the surrounding colonies.
3. Take a p200 tip, touch the colony surface and put the tip in the tube with the medium.
4. Close the tube (depending on the type of tube you want to leave the cap slightly open to allow air to come in) and incubate the tube at 37 °C overnight to allow bacterial growth.

The following day, the tubes containing the clonal culture will be centrifuged and the pellets re-suspended. Then they will be frozen in order to be used for the practical in week 7. Week 7 Subject Plasmid purification, digest, and separation of fragments through agar gel electrophoresis. Introduction This practice follows the one done in week 6, where competent cells were transformed, plated on agar and a colony was used to set a new culture. In this practical, a miniprep will be performed to purify the plasmid from the 3 mL culture in the previous practical. Following, the amount of plasmid will be used to digest with specific restriction enzymes (RE). The digested fragments will be separated on an agarose gel to determine the size and get information on the quality of the plasmid. After the run, you will get information on:

• The quality of the plasmid DNA you made
• The size of the whole plasmid, both in the circular and in the linearized form (remember a plasmid in vivo is always a closed circle of DNA)
• The size of the backbone (i.e. the common structure for that plasmid, which excludes the gene of interest)
• The size of the gene of interest often called insert

In this way, you will be sure that the plasmid you generated is correct and suitable for the downstream applications. Eventually, you will use the cut fragments for further applications. Content In this practical you will perform:

1. Plasmid purification by miniprep
2. Plasmid digest by RE
3. Agarose gel electrophoresis

1. Plasmid purification by miniprep Intro The mini-prep is a quick method for isolating small amounts of plasmid DNA (about 1-5 ?g/ml culture) and is often used to screen for recombinant plasmids after cloning experiments. The DNA is rapidly isolated from an overnight culture by a number of methods and in the same day may be digested with restriction enzymes and run out on an agarose gel. Portions of isolated plasmids will be digested with selected restriction enzymes in this practical. In this section, you will use the 3 mL culture (set from the colony you picked) to purify your plasmid. The procedure includes a first step of alkaline lysis (i.e. dissolution) of the bacterial cells, followed by the precipitation of genomic DNA (the main bacteria DNA, which has different chemical properties from the Plasmid DNA), and the ethanol-mediated purification of the plasmid. Protocol

1. From the  3 mL culture, put 1.5 mL in one Eppendorf tube. Set the remaining 1.5 mL aside.
2. Pellet the Eppendorf tube by spinning in a microcentrifuge at 13,000 rpm for 3 minutes
3. Remove and discard the supernatant using the p1000 pipette.
4. Put the remain 1.5 mL of the culture on top of the pelleted bacteria and spin the culture at 13,000 rpm for 3 minutes.
5. Remove and discard the supernatant.  Make sure you remove all the liquid as it will impair the purification of the plasmid.
6. Flick the bottom of the tube to soften the pellet. Resuspend the plasmid by adding 0.2 mL of P1 (resuspension buffer). Mix the pellet and P1 solution until resuspended. You should not observe clumps or unsuspended pellet.
7. Alkaline lysis. add 0.2ml of solution P2 (lysis buffer, 0.2M NaOH, 1% SDS) to the resuspended sample (this will lyse the cells and denature the genomic DNA) and mix gently by inverting the tube 10 times. Without delay move to step 7.
8. To precipitate the genomic DNA and bacteria debris from solution we change the alkaline pH back to neutral by addition of 0.2 mL solution P3 (neutralization buffer, 1.32M KOAc pH 5.0). Mix gently and leave on the bench at room temp for 5 min
9. Centrifuge the sample at top speed for 10 min to pellet the genomic DNA and bacteria debris.  The plasmid DNA will remain in the supernatant solution.
10. Ethanol precipitation. After the centrifugation you will end up with two phases, where the genomic DNA and the bacterial debris are in the lower phase and the plasmid DNA will be in the upper phase. With p200 carefully transfer the plasmid containing upper phase (about 0.4ml) into a clean labelled microfuge tube. You can discard the original tube containing the remnants of bacterial cell and genomic DNA.
11. Add 1ml of ice-cold ethanol to the plasmid-containing supernatant. Carefully mix with the p1000.
12. Incubate at room temperature for 5 min to precipitate the plasmid DNA.
13. Centrifuge the sample for 15 mins at top speed to pellet the precipitated DNA plasmid (it will be invisible at the bottom of the tube). Allow more people to group and centrifuge more samples at the same time. During this centrifugation prepare 1 mL of a mix made by 70% of ethanol and 30% of water, that you will use in step 15.
14. carefully pour off the supernatant. To do this, fold a piece of absorbing paper on the bench and pour the ethanol on it TOUCHING THE PAPER WITH THE EDGE OF THE EPPENDORF.
15. Add the 70% ethanol mix to the pelleted DNA and mix well (shake or vortex the tube)
16. Centrifuge the DNA sample as in step 13.
17. Pour away the ethanol as in step 14.
18. Put the empty tube with the open lid in the thermoblock set at 42 degrees for 5 minutes to completely dry the residues of ethanol (ethanol residues will impair downstream enzymatic reactions if left).
19. Add 50 µl of TE buffer, pH 8.0 to your DNA pellet and resuspend the plasmid pipetting up and down 10 times.

2. Plasmid digestion by RE Intro After the plasmid purification, you will use specific restriction enzymes to cut the gene of interest (COX2 gene) out of the plasmid and then run on agarose gel electrophoresis to check that the resulting fragments have the correct size. To do this you need to refer to the datasheet associated of the plasmid (see map below) and identify the restriction sites to be cut.  To cut the plasmid you will use HindIII and EcoRI enzymes. Here follows the map of the plasmid you are purifying. Can you localize the HindIII and EcoRI sites? Based on their relative positions, are you able to calculate the size of the resulting fragments?

• HindIII position: _____
• EcoRI position: _____
• Size of the two fragments:   ________ and ________

For each plasmid you will set 5 reactions, which will produce different and recognizable patterns on the gel:

This Report Writing Assessment has been solved by our Report Writing experts at My Uni Paper. Our Assignment Writing Experts are efficient to provide a fresh solution to this question. We are serving more than 10000+ Students in Australia, UK & US by helping them to score HD in their academics. Our Experts are well trained to follow all marking rubrics & referencing style.