DNA microarray method

1)            Set-up the following Pre-Hybridisation solution in a Coplin Jar and        incubate at65°C during the labeling incubation period to equilibrate. 20X SSC 8.75 ml 20% SDS 0.25 ml BSA (100 mg/ml) 5.0 ml H2O to 50.0 ml

2)            Label control and test genomic DNA as follows:- CONTROL TEST Genomic DNA ˜ 2 mg ˜ 2 mg Random Hexamers (3 mg/ml) 1 ml 1 ml H2O to 41.5 ml to 41.5 ml Heat at 95ºC for 5 minutes. Snap cool on ice and briefly centrifuge. 10X buffer 5 ml 5 ml dNTP's (5mM each dATP, dGTP & dTTP, 2mM dCTP) 1 ml 1 ml Cy-labelled dCTP 1.5 ml (Cy3) 1.5 ml (Cy5) Klenow fragment (10U/ml) 1 ml 1 ml Incubate at 37°C for 90 minutes.

3)          Incubate the microarray slide(s) in the Pre-Hybridisation solution for 20 minutes at65°C, beginning just before the end of the labelling reactions incubation time at37°C.

4)          Combine the control and test reactions and purify using the Qiagen MinElute PCR Purification kit, using a two step wash stage using 500 ml then 250 ml volumes of Buffer PE and eluting the labeled cDNA from the MinElute column with 14 ml H2O. The columns retain approximately 1 ml, so the final eluted volume will be 13 ml.

5)           Rinse the pre-hybridised microarray slides in H2O for 1 minute, then in isopropanol for 1 minute. Spin at 1500 rpm for 5 minutes to dry slides. Keep in covered slide box. 1 NICK DORRELL - LAST UPDATE FEBRUARY 2004

6)          Prepare the Hybridisation solution as follows: - Sample 13 ml H2O 26 ml 20X SSC 12 ml 2% SDS 9 ml Heat at 95ºC for 2 minutes. Allow to cool slowly at room temperature and centrifuge for 30 seconds. Add 2 x 20 ml H2O to the corners of the hybridisation chamber. Place a slide into the chamber. Place a LifterSlip™ glass coverslip (22 mm x 25 mm) over the array section on the slide using tweezers. Pipette the Hybridisation solution onto the slide at the top of the coverslip. Seal the chamber and incubate in a water bath at 65°C overnight.

7)         Prepare Wash solutions as follows: - Wash A (1X SSC 0.5% SDS) Wash B (0.06X SSC) 20X SSC 20 ml 2.4 ml 20% SDS 1 ml H2O to 400 ml to 800 ml Incubate Wash A solution at 65ºC overnight. Dispense 400 ml volumes into three glass slide washing dishes. Remove slide(s) from the hybridisation chambers and gently remove coverslip(s) by rinsing in Wash A. Place slide(s) in a slide rack and rinse with agitation for 5 minutes. Transfer slide(s) to a clean slide rack and rinse with agitation in Wash B(i) for 2 minutes, then in Wash B (ii) for a further 2 minutes. Spin at 1500 rpm for 5 minutes to dry slide(s).

8)      Scan slide(s) using Affymetrix 418 scanner and analyse data

（NICK DORRELL - LAST UPDATE FEBRUARY 2004）

Cloning Fact Sheet: questions and answers (2)

What animals have been cloned?

Scientists have been cloning animals for many years. In 1952, the first animal, a tadpole, was cloned. Before the creation of Dolly, the first mammal cloned from the cell of an adult animal, clones were created from embryonic cells. Since Dolly, researchers have cloned a number of large and small animals including sheep, goats, cows, mice, pigs, cats, rabbits, and a gaur. All these clones were created using nuclear transfer technology.

Hundreds of cloned animals exist today, but the number of different species is limited. Attempts at cloning certain species have been unsuccessful. Some species may be more resistant to somatic cell nuclear transfer than others. The process of stripping the nucleus from an egg cell and replacing it with the nucleus of a donor cell is a traumatic one, and improvements in cloning technologies may be needed before many species can be cloned successfully.

Can organs be cloned for use in transplants?

Scientists hope that one day therapeutic cloning can be used to generate tissues and organs for transplants. To do this, DNA would be extracted from the person in need of a transplant and inserted into an enucleated egg. After the egg containing the patient's DNA starts to divide, embryonic stem cells that can be transformed into any type of tissue would be harvested. The stem cells would be used to generate an organ or tissue that is a genetic match to the recipient. In theory, the cloned organ could then be transplanted into the patient without the risk of tissue rejection. If organs could be generated from cloned human embryos, the need for organ donation could be significantly reduced.

Many challenges must be overcome before "cloned organ" transplants become reality. More effective technologies for creating human embryos, harvesting stem cells, and producing organs from stem cells would have to be developed. In 2001, scientists with the biotechnology company Advanced Cell Technology (ACT) reported that they had cloned the first human embryos; however, the only embryo to survive the cloning process stopped developing after dividing into six cells. In February 2002, scientists with the same biotech company reported that they had successfully transplanted kidney-like organs into cows. The team of researchers created a cloned cow embryo by removing the DNA from an egg cell and then injecting the DNA from the skin cell of the donor cow's ear. Since little is known about manipulating embryonic stem cells from cows, the scientists let the cloned embryos develop into fetuses. The scientists then harvested fetal tissue from the clones and transplanted it into the donor cow. In the three months of observation following the transplant, no sign of immune rejection was observed in the transplant recipient.

Another potential application of cloning to organ transplants is the creation of genetically modified pigs from which organs suitable for human transplants could be harvested . The transplant of organs and tissues from animals to humans is called xenotransplantation.

Why pigs? Primates would be a closer match genetically to humans, but they are more difficult to clone and have a much lower rate of reproduction. Of the animal species that have been cloned successfully, pig tissues and organs are more similar to those of humans. To create a "knock-out" pig, scientists must inactivate the genes that cause the human immune system to reject an implanted pig organ. The genes are knocked out in individual cells, which are then used to create clones from which organs can be harvested. In 2002, a British biotechnology company reported that it was the first to produce "double knock-out" pigs that have been genetically engineered to lack both copies of a gene involved in transplant rejection. More research is needed to study the transplantation of organs from "knock-out" pigs to other animals.

What are the risks of cloning?

Reproductive cloning is expensive and highly inefficient. More than 90% of cloning attempts fail to produce viable offspring. More than 100 nuclear transfer procedures could be required to produce one viable clone. In addition to low success rates, cloned animals tend to have more compromised immune function and higher rates of infection, tumor growth, and other disorders. Japanese studies have shown that cloned mice live in poor health and die early. About a third of the cloned calves born alive have died young, and many of them were abnormally large. Many cloned animals have not lived long enough to generate good data about how clones age. Appearing healthy at a young age unfortunately is not a good indicator of long-term survival. Clones have been known to die mysteriously. For example, Australia's first cloned sheep appeared healthy and energetic on the day she died, and the results from her autopsy failed to determine a cause of death.

In 2002, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported that the genomes of cloned mice are compromised. In analyzing more than 10,000 liver and placenta cells of cloned mice, they discovered that about 4% of genes function abnormally. The abnormalities do not arise from mutations in the genes but from changes in the normal activation or expression of certain genes.

Problems also may result from programming errors in the genetic material from a donor cell. When an embryo is created from the union of a sperm and an egg, the embryo receives copies of most genes from both parents. A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a gene (either the maternal or paternal gene) is turned on. Defects in the genetic imprint of DNA from a single donor cell may lead to some of the developmental abnormalities of cloned embryos.

Should humans be cloned?

Physicians from the American Medical Association and scientists with the American Association for the Advancement of Science have issued formal public statements advising against human reproductive cloning. The U.S. Congress has considered the passage of legislation that could ban human cloning.

Due to the inefficiency of animal cloning (only about 1 or 2 viable offspring for every 100 experiments) and the lack of understanding about reproductive cloning, many scientists and physicians strongly believe that it would be unethical to attempt to clone humans. Not only do most attempts to clone mammals fail, about 30% of clones born alive are affected with "large-offspring syndrome" and other debilitating conditions. Several cloned animals have died prematurely from infections and other complications. The same problems would be expected in human cloning. In addition, scientists do not know how cloning could impact mental development. While factors such as intellect and mood may not be as important for a cow or a mouse, they are crucial for the development of healthy humans. With so many unknowns concerning reproductive cloning, the attempt to clone humans at this time is considered potentially dangerous and ethically irresponsible.

(Source: www.ornl.gov)

Cloning Fact Sheet: questions and answers (1)

Introduction

The possibility of human cloning, raised when Scottish scientists at Roslin Institute created the much-celebrated sheep "Dolly" (Nature 385, 810-13, 1997), aroused worldwide interest and concern because of its scientific and ethical implications. The feat, cited by Science magazine as the breakthrough of 1997, also generated uncertainty over the meaning of "cloning" --an umbrella term traditionally used by scientists to describe different processes for duplicating biological material.

What is cloning? Are there different types of cloning?

When the media report on cloning in the news, they are usually talking about only one type called reproductive cloning. There are different types of cloning however, and cloning technologies can be used for other purposes besides producing the genetic twin of another organism. A basic understanding of the different types of cloning is key to taking an informed stance on current public policy issues and making the best possible personal decisions. The following three types of cloning technologies will be discussed: (1) recombinant DNA technology or DNA cloning, (2) reproductive cloning, and (3) therapeutic cloning.

Recombinant DNA Technology or DNA Cloning

The terms "recombinant DNA technology," "DNA cloning," "molecular cloning," and "gene cloning" all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.

Scientists studying a particular gene often use bacterial plasmids to generate multiple copies of the same gene. Plasmids are self-replicating extra-chromosomal circular DNA molecules, distinct from the normal bacterial genome (see image to the right). Plasmids and other types of cloning vectors were used by Human Genome Project researchers to copy genes and other pieces of chromosomes to generate enough identical material for further study.

To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA.

Plasmids can carry up to 20,000 bp of foreign DNA. Besides bacterial plasmids, some other cloning vectors include viruses, bacteria artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Cosmids are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA and can be packaged in lambda phage particles for infection into E. coli cells. BACs utilize the naturally occurring F-factor plasmid found in E. coli to carry 100- to 300-kb DNA inserts. A YAC is a functional chromosome derived from yeast that can carry up to 1 MB of foreign DNA. Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.

Reproductive Cloning

Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.

Therapeutic Cloning

Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryo, which raises a variety of ethical concerns. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.

In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle less than 2/10,000th of an inch wide. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.

How can cloning technologies be used?

Recombinant DNA technology is important for learning about other related technologies, such as gene therapy, genetic engineering of organisms, and sequencing genomes. Gene therapy can be used to treat certain genetic conditions by introducing virus vectors that carry corrected copies of faulty genes into the cells of a host organism. Genes from different organisms that improve taste and nutritional value or provide resistance to particular types of disease can be used to genetically engineer food crops. With genome sequencing, fragments of chromosomal DNA must be inserted into different cloning vectors to generate fragments of an appropriate size for sequencing.

If the low success rates can be improved (Dolly was only one success out of 276 tries), reproductive cloning can be used to develop efficient ways to reliably reproduce animals with special qualities. For example, drug-producing animals or animals that have been genetically altered to serve as models for studying human disease could be mass produced.

Reproductive cloning also could be used to repopulate endangered animals or animals that are difficult to breed. In 2001, the first clone of an endangered wild animal was born, a wild ox called a gaur. The young gaur died from an infection about 48 hours after its birth. In 2001, scientists in Italy reported the successful cloning of a healthy baby mouflon, an endangered wild sheep. The cloned mouflon is living at a wildlife center in Sardinia. Other endangered species that are potential candidates for cloning include the African bongo antelope, the Sumatran tiger, and the giant panda. Cloning extinct animals presents a much greater challenge to scientists because the egg and the surrogate needed to create the cloned embryo would be of a species different from the clone.

Therapeutic cloning technology may some day be used in humans to produce whole organs from single cells or to produce healthy cells that can replace damaged cells in degenerative diseases such as Alzheimer's or Parkinson's. Much work still needs to be done before therapeutic cloning can become a realistic option for the treatment of disorders.

Oxidase test

Background: The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator.

Strains may either be oxidase positive (OX+) or negative (OX-).

OX+

OX+ normally means that the b

acterium contains cytochrome c oxidase and can therefore utilize oxygen for energy production with an electron transfer chain. Typically the Pseudomonadaceae are OX+[citation needed] Another example is the preliminary identification of Neisseria and Moraxella genera, which are both oxidase positive, Gram-negative diplococci.

Many Gram-negative spiral curved rods are also oxidase positive, which includes Helicobacter pylori, Vibrio cholera, and Campylobacter jejuni.

Also Legionella pneumophila is oxidase positive. A trick to remember the most medical relevant bacteria is: "VIce President CHeNEy MOstly LEads" (Vibrio, Pseudomonas, Campylobacter, Helicobacter, Neisseria, Moraxella, and Legionella, respectively).

OX-

OX- normally

means that the bacterium does not contain cytochrome c oxidase and therefore cannot utilize oxygen for energy production with an electron transfer chain. Typically Enterobacteriaceae are OX-.

Procedures

Wet each disk with about 4 inoculating loops of de-ionized water.

Use a loop to aseptically transfer a large mass of pure bacteria to the disk.

Observe the disk for up to 3 minutes. If the area of inoculation turns dark blue to maroon to almost black, then the result is positive. If a color change does not occur within three minutes, the result is negative.

Alternatively, live bacteria cultivated on trypticase soy agar plates may be prepared using sterile technique with a single-line streak inoculation. The inoculated plates are incubated at 37°C for 24–48 hours to establish colonies. Fresh bacterial preparations should be used. After colonies have grown on the media, two-to-three drops of the reagent DMPD is added to the surface of each organism to be tested.

A positive test (OX+) will result in a color change to pink, through maroon and into black, within 10–30 seconds.

A negative test (OX-) will result in a light pink coloration or absence of coloration.

Lipid Monolayer Assay Protocol

by Brian J. Peter and Matthew K. Higgins

Background

Lipid monolayers have been used for many years as templates for the formation of two-dimensional crystals of soluble proteins (reviewed in (1)) and, more recently, membrane proteins (2). The principle of the assay is that phospholipids on an aqueous droplet adopt a conformation in which the hydrophobic tails point towards the air while the hydrophilic head groups contact the solution. Proteins of interest interact with the head groups and are concentrated in a two-dimensional array. A hydrophobic electron microscope grid interacts with the lipid tails, allowing the monolayer to be removed from the droplet and studied in the electron microscope. The lipid monolayer simulates the inner leaflet of the plasma membrane, and can be used to reconstitute early stages of endocytosis. The formation of ordered clathrin assemblies can be observed using negative stain electron microscopy, and platinum shadowing can reveal the invagination of these coats. For examples, see references (3,4). A schematic of the technique and a gallery of images obtained with this technique can be viewed using the links, or at http://www2.mrc-lmb.cam.ac.uk/groups/hmm/epsin/EM/

Reagents

HKM buffer (25mM Hepes pH 7.4, 125mM potassium acetate, 5mM magnesium acetate, 1mM dithiothreitol).

Chloroform

methanol

Phosphatidylinositol and Phospatidylinositol-4,5-bisphosphate (Avanti polar lipids) and were dissolved to 1mg/ml in 3:1 chloroform: methanol. Cholesterol (also Avanti) was dissolved to 10mg/ml in Chloroform. Phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine (all from Sigma) were dissolved in chloroform to 10mg/ml. Lipid stocks were stored at -80C.

2% uranyl acetate (Biorad) with 0.0025% polyacrylic acid (Sigma) in water (see note 2)

Purified clathrin. Clathrin should be dialyzed into HKM buffer and centrifuged for 20 minutes at 100000 gmax (e.g., 45000 r.p.m. in a Beckman TLA100 rotor) immediately prior to use to remove aggregates.

Purified AP180, epsin, or other clathrin- and lipid-binding protein.

Equipment

Teflon block with 60 microliter wells allowing for side injection (see figure 1)

Carbon and collodion-coated gold electron microscopy grids (e.g., G204G from Agar Scientific, coated first with collodion or formvar, and then with a thin layer of evaporated carbon)

Humid chamber, or covered container with a wet paper towel inside

Forceps for handling EM grids—self-locking spring forceps are especially useful

Whatman filter paper or similar, for blotting EM grids.

Parafilm

Vacuum evaporator, 0.2mm diameter piece of platinum wire (TAAB Laboratories),1mm thick tungsten wire (also TAAB) (necessary for platinum shadowing)

Transmission electron microscope.

Procedure

1. Make up a lipid mixture containing 10% cholesterol, 40% PE, 40% PC and 10% PtdIns(4,5)P2 to a final concentration of 0.1mg/ml in a 19:1 mixture of chloroform: methanol (methanol is necessary to maintain PtdIns(4,5)P2 solubility). This mixture should be made on the day the monolayer is made. If stored, it should be stored under argon at –80 degrees in a glass vial with a glass or Teflon lid, for not longer than three days.

2. Arrange teflon block in humid chamber, and fill wells of teflon block with HKM buffer. Fill the wells with 40-60 microliters of buffer, such that the total volume in the well will be 60 microliters after injection of protein samples. See note 2.

3. Carefully pipet (or inject with Hamilton syringe) 1 microliter of lipid mixture on to the buffer in the well. As a negative control, inject pure chloroform without any lipid (this will test for lipid dependence of any structures seen, such as whether clathrin baskets form in solution instead of clathrin coats on the monolayer surface). See note 3.

4. Incubate at room temperature for 60 minutes. The chloroform should evaporate, leaving a monolayer of lipid on the surface of the buffer.

5. Carefully place one EM grid, carbon side down, onto the top of each buffer droplet. Grids should not be glow discharged before use as a hydrophobic carbon film is required to adhere to the hydrophobic lipid tails of the monolayer.

6. Gently inject proteins into the side injection well. Final protein concentrations in the well should be 0.5-2 micromolar for the AP180/epsin/adaptor protein, and 30-500 nanomolar for the clathrin. It is often useful to try several concentrations, to account for differences in protein activity.

7. Incubate 60 minutes at room temperature.

8. Prepare Uranyl acetate stain. Lay a fresh piece of Parafilm on the bench, and place two 30 microliter drops side by side on the Parafilm for each grid which will be stained. Lay out a piece of Whatman filter paper to blot buffer and stain from grids. Lay out a second piece of filter paper on which to set grids to air dry.

9. Gently inject approximately 30 microliters of buffer into the side injection port; this will raise the grid up above the surface of the teflon block. Immediately grab the grid with forceps and lift it vertically off of the droplet.

10. Blot the grid briefly by touching it to the filter paper, then touch it to the first stain droplet and blot immediately. Touch the grid to the second stain droplet, leave for 30 seconds, and blot briefly. This leaves a film of stain on the surface of the grid in which the protein is embedded. If the grids will be platinum shadowed, hold the grid to the filter paper for several seconds to ensure that the entire grid surface dries. Lay the grid on another piece of filter paper to dry.

11. For negative stain EM, grids can be examined in the EM immediately. They are also stable for several weeks, at least, at room temperature.

12. If platinum shadowing is required, set up the vacuum evaporator with a 2cm long piece of platinum wire coiled tightly round a piece of 1mm thick tungsten wire. Place the grids to be shadowed on a rotary platform at an angle of 10 to the line between the platform centre and the platinum coil. Create a vacuum in the evaporator. With a shield between the grids and the wire, turn on the current to the tungsten wire. When the platinum wire melts, remove the shield and the platinum will evaporate onto the grids. For rotary platinum shadowing, start rotation of the platform was immediately before removing the shield. For single angle shadowing, the platform can remain stationary during the evaporation. 1-2 minutes of evaporation is usually sufficient, but trials may need to be done to account for differences in evaporators.

Notes

This procedure will take approximately 3 hours. All reagents should be of the highest purity available, and buffers should be filtered before use.

1. The addition of polyacrylic acid helps to prevent the stain from precipitating and forming uranyl acetate crystals. But this is not essential, alternatively UrAc solutions can be clarified by filtration or centrifugation before use.

2. It is important to ensure that the surface of the droplet is either flat or slightly concave. Overfilling the teflon wells leads to a convex droplet surface, upon which the monolayer does not form properly. Also, filling the wells with too little volume (less than 35 microliters, or depending on the geometry of the block) can lead to an uneven surface near the side injection port.

3. Trace lipid contamination (e.g., PtdIns(4,5)P2) on the teflon block can result in misleading negative controls. Teflon block should be rinsed with hot water, then ethanol, and finally, soaked overnight in a mixture of chloroform/methanol to remove any protein or lipid residue. Hamilton syringes are also susceptible to trace lipid contamination.

References

1. Chiu W, Avila-Sakar AJ, Schmid MF. Electron crystallography of macromolecular periodic arrays on phospholipid monolayers. Adv Biophys. 1997;34:161-72.

2. Levy D, Mosser G, Lambert O, Moeck GS, Bald D, Rigaud JL. Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.
J Struct Biol. 1999 Aug;127(1):44-52

3. Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT. Curvature of clathrin-coated pits driven by epsin. Nature. 2002;419(6905):361-6.

4. Ford MG, Pearse BM, Higgins MK, Vallis Y, Owen DJ, Gibson A, Hopkins CR, Evans PR, McMahon HT. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science. 2001;291(5506):1051-5.

Source:

http://www2.mrc-lmb.cam.ac.uk/groups/hmm/techniqs/mono.html

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, U.K. CB2 2QH

If there are questions or comments, contact bpeter@mrc-lmb.cam.ac.uk or Harvey: hmm@mrc-lmb.cam.ac.uk

Lipid-Protein Overlay Assay (Fat Blotting)

Principle of the assay: This assay will test specific interactions between proteins and

immobilized lipids. It is essentially most similar to immunoblotting, with the added step of protein

binding preceding the antibody steps.

Special reagents and equipment:

Fatty Acid-Free (FAF) BSA Sigma Cat# A6003

Hybond C extra membrane Amersham Biosciences Cat # RPN2020E

Procedure:

1. Dilute lipids of interest in chloroform to equal concentration such that your desired amount

of lipid can be achieved in a total volume of 5 ul. *

2. Spot 5 ul of each dilution of lipid on to Hybond C extra membrane (mixed ester supported

nitrocellulose). **

3. Allow the membrane to dry at room temperature for at least 1 hr post-spotting. ***

4. Wet the membrane by floating on nano-purified water for 10 minutes, followed by

equilibration in buffer for 5 minutes (TBS-T (0.1% Tween-20)).

5. Block membrane in 3% FAF-BSA/TBS-T for 1 hr at room temperature. ****

6. Dilute your protein to 0.2 μg/ml in 3% FAF-BSA/TBS-T. Incubate membrane with protein

solution overnight at 4

o

C.*****.

7. Next morning: wash the membrane 6 times (5 minutes for each wash) with TBS-T.

8. Incubate with primary antibody in 3% FAF-BSA/TBS-T for 1 hr. at room temperature (do

not use nonfat dry milk, use the FAF-BSA throughout the entire procedure).******

9. Wash as in 7.

10. Incubate with secondary antibody in 3% FAF-BSA/TBS-T for 1 hr at room temperature.

11. Wash 12 times (5 minutes each wash) with TBS-T.

12. Visualize protein binding using enhanced chemiluminescence. Expose to film for 5

minutes for an initial trial and modify time as needed. .*******

Reference:

1. Stevenson, J.M., I.Y. Perera, and W.F. Boss, A phosphatidylinositol 4-kinase pleckstrin

homology domain that binds phosphatidylinositol 4-monophosphate. J Biol Chem, 1998.

273(35): p. 22761-7.

2. Dowler, S., et al., Identification of pleckstrin-homology-domain-containing proteins with novel

phosphoinositide-binding specificities. Biochem J, 2000. 351(Pt 1): p. 19-31.

3. Jones, J.A. and Y.A. Hannun, Tight Binding Inhibition of Protein Phosphatase-1 by

Phosphatidic Acid. SPECIFICITY OF INHIBITION BY THE PHOSPHOLIPID. J Biol Chem,

2002. 277(18): p. 15530-8. Jones JA, Hannun YA.Tight binding inhibition of protein phosphatase-1 by phosphatidic acid.

Specificity of inhibition by the phospholipid. J Biol Chem. 2002 May 3;277(18):15530-8. Epub

2002 Feb 20.

Author: Jeffrey Jones (modified by Bill Wu)

Your email: wub@musc.edu

Date: Dec-2009

Hop DNA Extraction Protocol

1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).

2. Assume 90% of mass is water weight.

3. Add 3.3 ml of buffer per gram of wet (16 ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65°C.

4. Transfer 900 μl into fresh tube

5. add 600 μl of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).

6. Centrifuge at 5000g for 10 minutes.

7. Transfer supernatant (800 μl) into new 2-ml tube.

8. Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).

9. Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.

10. Spin down for 10 min. at RT.

11. Add 500 μl wash buffer and incubate 10 min. at RT.

12. Carefully remove wash buffer. Don't lose DNA pellet!

13. Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.

14. Dry pellet at RT or 50°C to speed up.

15. Add 100 μl ddH2O to dissolve DNA.

16. Store at -20°C until needed.

17. Run electrophoresis for analysis.

Prepared solutions

Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 10 mg/ml of CTAB ( 200 mg per 20 ml buffer, final conc. = 1%) and 1 μl/ml 2-mercaptoethanol (20 μl to 20 ml buffer; final conc. = 0.1%).

Wash buffer 100 ml: 200 μl 5M NH4OAc (final conc. = 10 mM), 76.0 ml abs. ethanol (final conc. = 76%), and 23.8 ml of sterilized water.

Bioprotocols: Hop DNA Extraction Protocol

25 Dec 2010 ... Hop DNA Extraction Protocol. 1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount ...
bio888.blogspot.com/2010/12/hop-dna-extraction-protocol.html

Robust CTAB-activated charcoal protocol for plant DNA extraction
author：M KRIŽMAN - 2006
Dried hop cones were obtained from the experimental fields (yield 2005) .... Modification of a CTAB DNA extraction protocol for plants ...
fp.unud.ac.id/biotek/wp-content/uploads/biologisel/ekstraksi-dna.pdf

Isolation of plant DNA: A fast, inexpensive, and reliable method

author：P Guillemaut - 1992
Protocol. Isolation of plant DNA. DNA can be isolated from fresh, frozen, dried or lyophilized material without pretreatment of tissue. The procedure ...
www.springerlink.com/index/2656658377412530.pdf