Universitetet i oslo



NUTRIENTS and GENE REGULATION

[pic]

Study program for Nutrition

Department of Nutrition

Medical Faculty

University of Oslo

PROGRAM AND TIME SCHEDULE

|WEEK |44 |45 |46 |

|TOPIC |TOPIC 1 |TOPIC 2 |

| | | |

| | | |

| | | |

| | | |

| | | |

|Lectures |Laboratory course |Written exam |

|Start 1st of November 2010 |Start 22nd of November 2010 | |

|Time: 09:00-11:00 room 2180 |Time: 09:00 room 1130 |17th of December |

| | |Time: 09-14 |

|Lectures and self studies |Practical course that teaches you basic techniques |Rooms 2180 and 2183 |

|Course based on research |used in molecular biology. The techniques are used to| |

| |study gene regulation by nutrients. | |

|3 topics that will cover: | |5 hours |

|Key proteins and key transcription factors regulating |The topics in the lab course are based on the topics | |

|energy balance and insulin signalling pathways. The |of |The exam contains |

|themes will give insight into how metabolism of fat |SUBJECT BLOCK I. |questions from the |

|and sugar are closely interrelated and what factors | |lectures, the papers, and |

|interplay to regulate this metabolism. | |the lab course. |

| | | |

|Every topic lasts 1 week. | | |

|4-5 central scientific papers will be gone through. | | |

|Two teachers will give the lessons. | | |

|You will give an oral presentation week 49 from one of| | |

|these topics. No marks will be given, but you must | | |

|pass this before you can take the exam the 17th of | | |

|December! | | |

[pic]

[pic]

TOPIC 1:

Nuclear receptors in control of glucose and lipid homeostasis

Lecturer: Professor Hilde Nebb

TOPIC 2:

SREBP transcription factors - regulators of cholesterol and fatty acid metabolism.

Lecturer: Post.Doc. Fred Haugen

TOPIC 3:

Lipid droplet-associated proteins

Lecturer: Post.Doc. Knut Tomas Dalen

Responsible: Hilde Nebb/Knut Tomas Dalen

Teacher: Engineer Borghild Arntsen

TOPIC 1

NUCLEAR RECEPTORS IN CONTROL OF GLUCOSE AND LIPID HOMEOSTASIS

Professor Hilde Nebb

It is known that fat plays an important role for development of obesity, diabetes type II, coronary heart disease and cancer. Both heritage and environmental factors are crucial for the predisposition of these diseases, and the diet is a pivotal factor. The food contributes with nutrients used for construction and maintenance. Transcription factors are messengers that convey signals between intracellular signals and the genes. Nuclear receptors such as Liver X Receptor (LXR), Peroxisomal Proliferator-Activated Receptor (PPAR) and Retinoid X Receptor (RXR) are such cellular messengers, conveying signals from the nutrients cholesterol compounds, fatty acids and vitamin A, respectively.

The nuclear receptor superfamily consists of about 48 receptors (in man), including steroid hormone receptors and receptors for vitamin A, thyroid hormone and vitamin D. This group also includes receptors for both saturated and unsaturated fatty acids, cholesterol metabolites like oxysterols and bile acids, in addition to orphan receptors where the physiological ligand is still not known.

This family of transcription factors has a crucial role in regulation of gene expression, and mediates a communication between extracellular signals and transcriptional responses. They recognize short DNA sequences on target genes that are specific for the receptors.

These DNA motifs are called DNA response elements (RE). Other names are hormone response elements (HRE) or cis-elements. The motifs are localized upstream at the target genes. A transcriptional activation of the gene occurs when the ligand and the receptor make a complex that bind to the specific HRE.

Several nuclear receptors participate in central metabolic pathways in glucose and lipid homeostasis. The glucose and lipid homeostasis is a delicate balance between food intake, synthesis and degradation. The liver, muscle and adipose tissue are main organs in this balance.

Topic I discusses some of these regulatory pathways where nuclear receptors play a key role, in order to elucidate the influence of these receptors on glucose and lipid homeostasis – alone or in an interaction.

Literature:

1. Evans RM The nuclear receptor superfamily: A rosetta stone for physiology

Mol Endo. 2005. 19(6):1429-1438



2. Lonard DM, Lanz RB nad O’Malley BW. Nuclear receptor coregulators and human disease. Endocrine Reviews. 2007. 28(5):575-587



3. Chawla, A. et al. Nuclear receptors and lipid physiology: opening the x-files. Science. 2001, 294:1866-1870



4. Steffensen, KR and Gustafsson, JA. Putative metabolic effects of the liver X receptor. Diabetes. 2004,Feb;53, suppl 1:S36-42



5. Mitro N et al. The nuclear receptor LXR is a glucose sensor. Nature. 2007.

455:219



6. Dalen, KT et al. Expression of the insulin-responsive glucose transporter GLUT4 in adipocytes is dependent on liver X receptor alpha. J Biol. Chem. 2003. 278(48):48283-91.



Suggested further reading:

1. Mangelsdorf, DJ and Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995, 83(6):841-50.

2. Baranowski M (review) Biological roles of liver X receptors (2008), 59, Suppl 7,31-55.

3. Wang YX (review) PPARs: diverse regulators in energy metabolism and metabolic diseases. Cell Res. 2010 Feb;20(2):124-37



TOPIC 2

SREBP transcription factors - regulators

of cholesterol AND fatty acid metabolism

Fred Haugen, Dr.philos

The sterol regulatory element-binding protein (SREBP) transcription factors are critical regulators of cholesterol/lipid homeostasis, which act by controlling the expression of many cholesterogenic and lipogenic genes. We will look into scientific work that indicates a role for polyunsaturated fatty acids in regulation of gene expression.

A summary of SREBP regulation will be given and several articles will be presented in depth.

Literature:

Hannah et al.

Unsaturated Fatty Acids Down-regulate SREBP Isoforms 1a and 1c by Two Mechanisms in HEK-293 Cells.

J. Biol. Chem., Vol. 276, Issue 6, 4365-4372, February 9, 2001

Takeuchi et al.

Polyunsaturated fatty acids selectively suppress sterol regulatory element-binding protein-1 through proteolytic processing and autoloop regulatory circuit.

J Biol Chem. 2010 Apr 9;285(15):11681-91.

Gong et al.

Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake.

Cell Metab. 2006 Jan;3(1):15-24.

Radhakrishnan et al. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig.

Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6511-8. Epub 2007 Apr 11.

Najafi-Shoushtari et al.

MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis.

Science. 2010 Jun 18;328(5985):1566-9.

TOPIC 3

LIPID DROPLET-ASSOCIATED PROTEINS

Knut Tomas Dalen, Dr. philos

Lipid droplets (LDs) are found ubiquitously; in bacteria, yeast, plants and most eukaryotic cells studied. They exist in multiple sizes, ranging from droplets invisible to the light microscope to LDs that occupy most of the cellular cytoplasm (200 ml) into the incubator.

Recepies:

[pic]

Day 2 Plasmid isolation (mini prep.)

[pic]

Task 1: Isolation of a plasmid containing YFP or YFP-Plin2

Bacteria reproduce rapidly. Under optimal conditions (when grown at 37(C and in abundance of nutrients), E coli divide every ~20 minutes. This property can be used for production of large amounts of a plasmid over night. Each of the newly formed bacteria contains one or more copies of the plasmid. Plasmids used in laboratories are usually high-copy plasmids, and designed to multiply to several hundred copies in each single E. coli. This ensures a high yield of isolate plasmids.

To ensures that only bacteria containing the plasmid survive, and to increase the number of plasmids in each bacteria, the bacteria are grown in the presence of an antibiotic (often ampicillin or kanamycin). The use of antibiotics has also the advantage that it limits the risk of contamination with other bacteria.

Today, we will isolate the two different plasmids that we transformed into the bacteria at day 1. Different volumes of bacteria culture is used, depending on the yield needed. 5 ml culture is normally used for a mini prep (expected yield 3-20 µg), 25 ml culture medium is used for a midi prep (yield 45-100 µg) while 100-200 ml medium is used for a more time-consuming, large-scale maxi prep (yield 300-500 µg).

(We will perform midi prep isolation of plasmid).

Protocol - Preparing a bacterial culture

(this step is performed by the lab teacher the night before).

Mini-prep isolation of DNA: Take a sterile tube with round bottom and add 2-5 ml autoclaved LB-medium (LB-medium is a nutritious medium for maximum bacterial growth) containing ampicillin (100 µg/ml). Pick a colony from the plate of transformed bacteria and dissolve it in the medium. Incubate the tube in the shaker at 37°C over night.

Maxi and mini prep plasmid isolation: Set up a small culture as described above in the morning, and transfer 1-2 ml of the culture into a larger volume of LB medium in the end of the day. Use 40 ml LB-medium for a midi prep and 250-500 ml for a maxi prep.

Protocol - Mini, Midi and Maxi Plasmind isolation

Jet-Star Plasmid Purification System

Follow the protocol given by the manufacturer.

You should follow the directions given for midi plasmid isolation.

Preparations:

Dissolve the lyophilized RNase A in solution E1 as recommended.

Column equilibration

1. Column Equilibration Columns are equilibrated before the cleared lysate is prepared by applying solution E4. The flow of solution starts automatically. Allow the column to empty by gravity flow. Do not force out remaining solution.

Solution E4 / Mini: 2 ml

Midi: 10 ml

Maxi: 30 ml

2. Harvesting Bacterial Cells E. coli cells are pelleted by centrifugation. Remove all traces of medium carefully.

3. Cell Resuspending Add solution E1 to the pellet and resuspend the cells until the suspension is homogeneous.

Solution E1 / Mini: 0.4 ml

Midi: 4.0 ml

Maxi: 10.0 ml

4. Cell Lysis Add solution E2 and mix gently, but thoroughly by inverting until the lysate appears to be homogeneous. Do not vortex! Incubate at room temp. for 5 min.

Solution E2 / Mini: 0.4 ml

Midi: 4.0 ml

Maxi: 10.0 ml

5. Neutralization Add solution E3 and mix immediately by multiple inverting until a homogeneous suspension is obtained. NO REMAINDERS OF THE VISCOUS MATTER THAT APPEARED AFTER CELL LYSIS (Step 4) MUST BE LEFT! Do not vortex! Centrifuge the mixture at room temperature

and 12.000 x g for 10 min.

Solution E3 / Mini: 0.4 ml

Midi: 4.0 ml

Maxi: 10.0 ml

6. Column Loading Apply the supernatant from step 5 to the equilibrated JETSTAR column. Allow the lysate to run by gravity flow.

7. Column Washing Wash the column with solution E5 twice (Mini and Midi) or once when Maxi is used. Allow the column for each wash to empty by gravity flow.

Solution E5 / Mini: 2 x 2.5 ml

Midi: 2 x 10.0 ml

Maxi: 1 x 60.0 ml

8. Plasmid Elution Elute the DNA with solution E6. Do not force out remaining solution.

Solution E6 / Mini: 0.9 ml

Midi: 5.0 ml

Maxi: 15.0 ml

9. Plasmid Precipitation Precipitate the DNA with 0.7 volumes of isopropanol (Mini: 0.63 ml, Midi: 3.5 ml or Maxi: 10.5 ml). Centrifuge at 4°C and 12.000 x g for 30 min. Wash the plasmid DNA with 70% ethanol and recentrifuge. Air dry the pellet for 10 min, and redissolve the DNA in a suitable volume of buffer (i.e. 10 mM Tris-Cl, pH 8.0, TE buffer or water).

Task 2: Measure light absorbance by DNA and calculate DNA concentration

Spectrophotometric DNA and RNA quantification is based on absorption of light at specific wavelengths by the conjugated ring structures present in nucleic acids. Nucleic acids absorb light with a maximal absorption peak at 260 nm. An optical density (OD) of 1 at 260 nm corresponds to 50 μg/ml double-stranded DNA or 40 μg/ml RNA or single-stranded DNA. The linear range for measuring nucleic acids at OD260 is 0,1-1,0.

Other biological molecules and compounds frequently used in the preparation of nucleic acids absorb light in the 230-320 nm range. The presence of such impurities in the measured sample can result in over-estimation of the nucleic acids concentration. When quantifying nucleic acids in solution, it is therefore common to measure the absorption at 260 nm and 280 nm simultaneously, and use the calculated OD260/280 ratio as a quick estimate of the samples purity.

Phenol absorbs with a peak at 270nm, with a A260/280 ratio of 2. Proteins absorb light with a maximal peak at 280 nm, but the efficiency is low. The OD A260/280 ratio can be used to determine the amount of contaminating DNA in a protein sample, where the ratio between OD260 and OD280 is an indicator of the sample’s purity. However, contradictionary to a common believe, the OD280/260 ratio does not give a good estimate of protein contamination in a DNA sample, as the amount of protein must be high before it absorbs significantly at OD280.

Pure DNA should have an OD260/280 ratio around 1.8, while RNA should have a ratio close to 2.0. If the numbers above are lower, the sample could be contaminated by a significant amount of proteins (or ethanol).

Protocol - Measure absorbance using a spectrophotometer:

Plastic absorbs light in this region, and quartz cuvettes must be used for measurements.

1. Dilute the sample 100 x by adding 5 μl plasmid solution to 495 (l water and 250 x by adding 2 μl to 498 µl water.

2. Prepare a blind sample, containing water only.

3. Vortex the sample and spin down.

4. Wash the cuvette thoroughly with water; remove all traces of water with a pipette.

5. Add 500 μl of the blind sample to the cuvette and make a baseline by pushing "Set ref"

6. Remove all traces of the blind sample and add the sample to be measured.

7. Measure OD at 260 nm and 280 nm.

8. Calculate the OD260/280 ratio.

Protocol - Calculation of DNA concentration:

[pic]

Day 3 Restriction of DNA, agaroge gel electrophoresis and culturing of CHO cells

[pic]

Task 1: Cut out the Plin2 cDNA from the pEYFP-Plin2 plasmid using restriction enzymes BglII and KpnI

Restriction enzymes catalyze a break of double-stranded DNA at specific nucleotide recognition sites. Each enzyme recognizes a clearly defined DNA nucleotide sequence ranging in 3 to 8 nucleotides in length; often palindromic. The majority of restriction enzymes make a staggered symmetrical cut in DNA and generate sticky ends. Others produce blunt ends. A few restriction enzymes cut the DNA several nucleotides away from their recognition site.

Restriction enzymes are frequently used in cloning, where a specific DNA sequence is digested out from one DNA-source, for transfer into a new DNA-source using an enzyme that connects DNA (ligase). Restriction enzymes are e.g. used to transfer PCR products to a plasmid or move a cDNA sequence from one plasmid to another. If the nucleic acid sequence of a DNA source is known, computer programs is often used to find proper restriction enzymes to cut the DNA at the desired site(s).

Restriction enzymes are isolated from numerous organisms, living in quite different environments. Every restriction enzyme therefore has individual salt, pH and temperature requirements for optimal catalytic activity. Some enzymes are quite specific in their requirements, whereas other works well over a broader range of salt and pH in the reaction solution. Every restriction enzyme is therefore supplied with a buffer that provides optimal catalytical activity. If two enzymes is used at the same time, it is important to use a buffer that works well for both enzymes. For some combinations of enzymes, it is not possible to find a common buffer, and the DNA has to be cut two times.

In our experiment, we will use restriction enzymes to test if we have purified the correct plasmids. We will use the same restriction enzymes that were used to clone the Plin2 cDNA (1290 bp) into the YFP-containing vector (pEYFP-C1; ~4.7 kb). The Plin2 cDNA was inserted using the restriction enzyme KpnI.

Protocol - DNA restriction enzyme cutting:

Cut both of the plasmids you isolated on day 2 (the pEYFP-C1 vector and the pEYFP-Plin2 vector).

1. Pipette out all reagents below to eppendorf tubes. Let the pipette tip only just pierce the liquid surface, to avoid getting any liquid outside the tip.

|Amount: |Reagent: |

|x μl = 3 μg pEYFP-Plin2/ pEYFP-C1 plasmid DNA |DNA |

|3 μl |10 x L buffer |

|0.3 μl |100 x BSA |

|0.5 μl |KpnI 10 U/ μl |

|x µl (up to 30 μl) |H2O |

|Total: 30 μl | |

2. Mix thoroughly by vortexing or tapping on the tube.

3. Centrifuge briefly at 1000 rpm for some seconds to spin down the solution.

4. Incubate at 37°C for >1 hour.

Task 2: DNA separation by agarose gel electrophoresis

In order to determine the size of the DNA fragments produced after the restriction cutting, we use a method that separates DNA molecules based on size; agarose gel electrophoresis. The phosphate groups of DNA are negatively charged at neutral pH, and the electric charge of a DNA fragment is determined by the number of nucleotides only. In an electric field, DNA molecules will migrate towards the positive pole.

The DNA fragments are separated by size in a matrix made of agarose. The pore size in the matrix is determined by the agarose concentration and can be adjusted to optimize separation of small or large DNA molecules. The agarose gel network slows down large DNA fragments more than small, and hence, small fragments will migrate faster through the gel. Due to their difference in migration speed, the DNA fragments will over time be separated by size. Usually 0.5 % agarose is used to separate large fragments (>1000 bp), whereas up to 3% might be used to separate smaller DNA fragments (50-1000 bp). (Circular DNA, such uncut plasmids, will migrate differently, since the molecule will twist to form different helix structures.).

[pic]

DNA is invisible to the human eye. In order to see the DNA, we add substances that intercalates between the bases in DNA to the gel matrix; such as Ethidium bromide or GelRed. Both of these substances emit fluorescence when bound to DNA and exposed to UV light, and the DNA is then visible as bright bands. A standard (or ladder) containing DNA fragments of known size (100 bp to 12 kbp) is always run along with the samples.

[pic]

Protocol – Generation of an agarose gel

[pic]

1. Measure up 50 ml running buffer (1xTAE buffer) using a cylinder and pour it into a conical flask.

2. Weigh up agarose powder and transfer to the flask.

3. Cover the flask loosely and heat until the solution boils in the microwave to dissolve the agarose crystals. Don’t let it boil over!

4. Take out the solution every now and then to check if the agarose crystals are dissolved. The solution is ready when it is homogenous and clear. Do not get burned, use a cloth!

5. Cool down the solution on a water bath until ca 55(C.

6. Add GelRed (1 µl for each 100 ml agarose solution) using a pipette and swirl the flask.

7. Pour the solution carefully into the mould. Remove any bubbles with a pipette tip.

8. Place the comb at the top of the gel mould.

9. Let the gel stand at room temperature for >30 minutes.

Protocol - Applying DNA samples to the gel

1. Mix the digested samples with loading buffer according to the table below. In addition, make samples with non digested pEYFP-C1 and pEYFP-Plin2 plasmids.

| |Tube 1 |Tube 2 |Tube 3 |Tube 4 |Tube 5 |

| |Std. |pEYFP-C1 (not |pEYFP-Plin2 (not |pEYFP-C1 |pEYFP-Plin2 |

| |(1kb+) |digested) |digested) |(digested) |(digested) |

|DNA sample |1 µl |X µl (1 µg) |X µl (1 µg) |10 µl |10 µl |

|6 xLoading |2 µl |2 µl |2 µl |2 µl |2 µl |

|1 x TE Buffer |8.0 µl |10- X µl |10- X µl |- |- |

|Total volume |12.0 µl |12.0 µl |12.0 µl |12.0 µl |12.0 µl |

2. Remove the comb from the gel and put the gel (together with the slide) in an electrophoresis chamber containing 1x TAE buffer. Carefully pipette each sample into separate wells in the gel. The loading buffer contains glycerol, which ensures that the sample sinks down into the well, and colour to make it easier to se the sample while loading. During the electrophoresis, the colour might also be used to estimate the front of the samples, as the cyan colour will migrate with the same speed as small DNA fragments (20 minutes).

Tips for bacterial work:

Different bacteria may be present everywhere. Use of ampicillin will kill all the bacteria that don’t have an ampicillin resistance gene. In order to avoid unwanted bacteria in the samples, please follow these rules:

i) Use sterile disposable equipment only. Autoclave equipment before use.

ii) Flame all covers and openings.

Resistant bacteria and antibiotics may be harmful. Remember always to use lab coat and gloves.

Calculation:

Use the measured absorption at OD260 and OD280 to calculate:

• The amount of nucleic acid in the sample (use the formula below).

• Estimate the samples purity.

5’ 3’ mRNA

RT step

cDNA

synthesis

5’ Reverse primer

3’ 5’ cDNA strand

Cycles

Rn Amplification plot logarithmic Y axis

OD260 x 40 x 100 / 1000 = μg/μl _________________

OD = 1 → RNA = 40 μg/ml

Dilution factor

convert ml to μl

Precautions:

RNA is easily degraded by the enzyme RNase. This enzyme is present almost everywhere – on our hands, clothes, bottles, etc. Therefore, working with RNA is very demanding. Please take these precautions:

i) Use lab coat and gloves, and change gloves as required.

ii) Wash the bench before use, and use a new bench cover.

iii) Use sterile disposable equipment only.

iv) RNases are not destroyed by autoclavation. Water should be treated with DEPC to deactivate RNases.

v) RNA must be stored at -70°C and thawed on ice only shortly before use

10 X Transfer Buffer

25mM TrisHCl

190mM Glycin

H2O (MQ) ad 1l

10 X Running Buffer, pH 7,4

96 mM (30 g) TrisBase

1,9 M (144 g) Glycin

10 % (10 g) SDS

H2O (MQ) ad 1l

Exercise:

Find out why antibiotics is removed from the culture medium

the day prior too, and during transfection?

Restriction Enzyme Cleaving site

Bgl II A GATC T

T CTGA T

Kpn I G GATC C

C CATG G

Sma I CCC GGG

GGG CCC

Xma I C CCGG G

G GGCC C

Purpose of today’s lab work (day 7):

Task 1: Run SDS-PAGE.

Task2: Electrotransfer (blotting)

Task3: Block membrane, and incubate with primary Ab – immunoblotting day 1

3’ 5’ cDNA

Purpose of today’s lab work (day 6):

Task 1: Measure protein concentration in the samples.

Task2: Dilute samples for Western blotting.

Exercise:

Again, compare the cells receiving different stimulation medium

Can you see a difference?

Can you see a difference among non-transfected cells, cells transfected with YFP or YFP-Plin2?

Exercise:

Compare the cells receiving different stimulation medium

Can you see a difference?

Can you see a difference among non-transfected cells, cells transfected with YFP or YFP-Plin2?

Special note:

• Formaldehyde is carcinogenic and may cause allergic reactions. Use gloves when handling!

Purpose of today’s lab work (day 5):

Task 1: Harvest one dish for protein extraction (dish A)

Task 2: Harvest one dish for RNA extraction (dish B)

Task 3: Fix and color one dish for (dish C)

5’ Forward primer

Preparation:

Calculate the amount of medium and stock solutions needed

Stimulate with OA-BSA after transfection

Stimulate with BSA after transfection

Purpose of today’s lab work (day 4):

Task 1: Transfect CHO cells with the isolated plasmids

Task 2: Stimulate the transfected CHO cells with fatty acids

Cell culturing tasks:

1: Trypsinate CHO cells.

2: Count the number of cells in your sample.

3: Seed CHO cells into dishes for later experiment.

Exercise:

Use the migration pattern of the 1 kb+ molecular ladder to estimate the size of the DNA fragments in your sample. Use this information to determine if the plasmids you have purified are likely to be the correct plasmids.

Some of the DNA fragments are brighter than the others. Why do the intensities of the DNA fragments differ?

Special notes:

• Substances that bind to and intercalates in DNA should be considered as carcinogenic until tested otherwise. GelRed is tested to be little carcinogenic and will be used in this lab course. Ethidium bromide is known to generate DNA mutations.

• Exposure to UV light generate DNA damage and should be avoided. Wear appropriate protecting clothing to protect eyes and skin (gloves, glasses).

Preparation:

Before you make the gel:

Determine the percentage of agarose that should use for optimal separation of the sample (based on the known size of the DNA fragments generated by the restriction digestion reaction).

Purpose of today’s lab work (day 3):

Task 1: Digestion of the plasmids purified in day 2.

Task 2: Separation of the digested plasmids by agarose gel electrophoresis.

Task 3: Count and seed CHO cells to be transfected on day 4.

Purpose of today’s lab work (day1):

Task 1: Transfer two different plasmids into E.coli bacteria using heat-shock

transformation.

Purpose of today’s lab work (day 2):

Task 1: Use E.coli bacteria as a factory to multiply plasmids transformed at day 1,

and isolate and purify these plasmids.

Task 2: Determine the amount of isolated plasmids.

5’ 3’ complementary

PCR

cycle 1

3’ 5’

5’ 3’ amplification

PCR

cycle 2

and later

SYBR Green exhibits weak fluorescence in solution, but strong fluorescence upon binding to double-stranded DNA

When they are both attached to the TaqMan probe, the quencher (orange) inhibits fluorescence from the reporter dye (blue).

When reporter dye is cleaved off the probe and separated from the quencher, it emits fluorescence.

TaqMan probe

Precautions:

RNA is easily degraded by the enzyme RNase. This enzyme is present almost everywhere – on our hands, clothes, bottles, etc. Therefore, working with RNA is very demanding. Please take these precautions:

vi) Use lab coat and gloves, and change gloves as required.

vii) Wash the bench before use, and use a new bench cover.

viii) Use sterile disposable equipment only.

ix) RNases are not destroyed by autoclavation. Water should be treated with DEPC to deactivate RNases.

x) RNA must be stored at -70°C and thawed on ice only shortly before use

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download