Cytokines and growth factors array

CYTOKINES & GROWTH FACTORS RESEARCH BIOCHIPS

Randox supplies biochips for all your cytokines & growth factors research. Biochips offer multiplexing; all analytes are measured simultaneously from one sample. Biochip array technology brings analytical clinical standards to research. Randox biochips are ready-to-use on the evidence investigator™ complete with controls and calibrators.

Cytokines and growth factors are proteins secreted primarily by immune system cells such as leucocytes. They are a means of intercellular communication with both immune and non-immune system cells. Upon binding to a cell membrane receptor, cytokines initiate a signal transduction cascade in their target cell. Cytokines form a complex web of interactions since:

Different cytokines can have similar effects.

Cells interact with a variety of cytokines.

Cytokines may affect the activity and expression of other cytokines.

evidence investigator
The evidence investigator is a compact bench-top Biochip Array Technology analyser for the simultaneous analysis of a panel of tests in a single sample. The Clinical standards for research page gives an overview of Biochip Array Technology.
The cytokines and growth factors array of tests are presented on a single biochip. All twelve tests are performed simultaneously using just one assay diluent, one panel-specific conjugate solution and one signal reagent.
Cytokines and growth factors array test menu
Interleukin-1 alpha (IL-1a)
Interleukin-1 beta (IL-1b)
Interleukin-2 (IL-2)
Interleukin-4 (IL-4)
Interleukin-6 (IL-6)
Interleukin-8 (IL-8)
Interleukin-10 (IL-10)
Interleukin-12 (IL-12) - in development
Vascular Endothelial Growth Factor (VEGF)
Tumour Necrosis Factor-alpha (TNF-a)
Interferon-gamma (IFN-g)
Epidermal Growth Factor (EGF)
Monocyte Chemotactic Protein-1 (MCP-1)
For more detailed information please e-mail: marketing@randox.com

Details: CYTOKINES & GROWTH FACTORS RESEARCH BIOCHIPS

Microbiology by Prescott-EBook

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The Cell - A Molecular Approach-E Book, The Cell - A Molecular Approach Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000

 

 

 

 

The Cell - A Molecular Approach

Cooper, Geoffrey M.

Sunderland (MA): Sinauer Associates, Inc. ; c2000

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Approved Lists of Bacterial Names-Ebook

The Approved Lists of Bacterial Names was published in the International Journal of Systematic Bacteriology (IJSB 30: 225-420, 1980) and reprinted in book form to provide for the requirements of the Bacteriological Code (1976 Revision) in initiating a new starting date for bacterial nomenclature, 1 January 1980.

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TA cloning or Subcloning of PCR Products Protocol

TA Subcloning of PCR Products

This procedure is adapted from D. Marchuk, M. Drumm, A. Saulino, and F.S. Collins Nuc. Acids. Res. (1991) 19:1154.

CONSTRUCTION OF T-VECTOR

1. suspend 10ug pUC 19 in:
   • 4.0ul 10X reaction buffer (we use Bo. Mann. buffer A)
   • 2.0ul (20U) Sma I
   • X ul dwater to a total vol. of 40ul
     Incubate at 30 (not 37) degrees for 1 hour. This is easier if done in a 0.4ml tube in a thermal cycler.
2. Heat to 70 degrees for 15 min. to kill the enzyme
3. Bring to 100ul w/ water (add 60ul).
4. Extract w/ phenol, phenol/chloroform and then chloroform.
5. add 9ul 3M sodium acetate.
6. ppt. in ETOH, wash with 70% ETOH (be careful with the pellet!).
7. Dry in spin vac at room temp (do not use heater!).

********************T-TAILING THE VECTOR******************

At this point, it is assumed that there has been 80% recovery of the cut plasmid DNA.

1. Resuspend the plasmid DNA in 63ul water (conc approx. 130ng/ul)
2. To the resuspended plasmid add:
   • 10ul 10X PCR buffer (standard cetus stuff, no MgCl)
   • 20ul 10mM dTTP [2mM final]
   • 6ul 25mM MgCl2 [1.5mM final]
   • 1ul Taq polymerase (Cetus amplitaq 5U/ul)
   • ______
   • 100ul total volume.
3. Incubate for 3 hours at 70 degrees C.
4. Extract with Phenol, Phenol/chloroform, chloroform.
5. Extract twice with ether (so I'm paranoid!)
6. add 75ul 2M **ammonium** acetate (assuming 75ul recovery from extractions).
7. Add 150ul isopropanol. Spin 20mins in microfuge at full speed at 4 degrees.
8. Wash with 70% ETOH 9 Dry pellet in spin vac and store at -20 degrees until use.

TREATMENT THE PCR PRODUCTS

" If you can see it, you can clone it".

1. Add an equal volume of chloroform (*NO* IAA) to the PCR reaction and spin 1-2 minutes in microfuge at RT.
2. Remove the oil which is now on the ****BOTTOM***.
3. Spin again for two minutes and remove the last little bit of oil from the bottom. You will know when you have gotten it all when you see the interface in the pipette tip. It is important that all the oil be removed otherwise subsequent procedures will be very difficult.
4. Add 100ul 4M ammonium acetate, vortex, and then add 200ul isopropanol.
5. Centrifuge 20min at 4 degrees, wash in 70% ETOH.
6. Dry in speed vac.
7. Resuspend the DNA in 8-10ul TE, add loading buffer and load onto a 4% Nusieve (TAE) agarose gel. Run until the desired band is well separated. The more DNA in the band, the easier it is to subclone.
8. Cut out the band. Minimize the exposure of the gel (and you!) to short wave UV

LIGATION OF PCR PRODUCTS TO T-VECTOR

1. Heat the gel containing the PCR fragment to 65C for 10 minutes, place in a 37C water bath or block and add to a separate tube (also at 37C):
   • 10 ul gel
   • 4ul 5X ligase buffer (commercial buffer that comes with BRL T4 ligase)
   • 4ul water
   • 1ul vector (25-50ng)
   • 1ul ligase
   • Incubate at 12C overnight.
2. Heat the mixture to 68 degrees for 5 minutes and add 100ul water.
3. Extract with phenol, phenol/chloroform, and chloroform. These steps are to remove the agarose.
4. Add 10ul 3M NaAcetate and precipitate with ethanol.
5. Wash the pellet in 70% ETOH, dry in the speed-vac. Resuspend in 5ul of water just prior to transformation.

*Transformation - We usually use electroporation into XL-1 blue cells. You need cells that can achieve at least 1 x 10^7 transformants per ug of DNA if a CaCl based protocol is used.

*Storage: The T-vector should be stored at -20C at all times. When stored in dry form, the T-overhangs will last longer (I don't know how long yet). In solution, it lasts at least a couple of weeks at - 20C.

*Enzymes - The batch of SmaI that is used is particularly critical. Some are contaminated with an endonuclease that removes a few bases from the cloning site. The batch of smaI should be checked before it is used to cut vector for cloning purposes. If bluescript is used, EcoRV can be substituted for sma I.

txpljfg@uabcvsr.cvsr.uab.edu

Cloning of Blunt-end PCR Fragments Protocol

Direct Cloning of Blunt-end PCR Fragments

BioTechniques 13:613

• Phenol extract the PCR product
• Ethanol precipitate
• Treat for 1hr at 37C with 10 units of T4 DNA polynucleotide kinase and 10 units T4 DNA polymerase I(NEB) in a 100ul reaction volume containing 50mM Tris-HCl pH7.5, 10mM MgCl2, 1mM DTT, 50 ug/ml BSA, 1mM ATP, 200uM each dNTP.

I run the entire thing out on a 1.3% agarose TAE gel, cut a trough in front of the band, pour in some 0.7% LMP agarose(BRL), run the product into it and excise.

The PCR product in the LMP can be used for ligations directly, without purification. The ligations take place at room temp on the benchtop. I prepare the vector with minimal digestion (~2hr) then treat it with shrimp alkaline phosphatase(USB). I usually prepare a stock of this vector to have on hand, so I know it is good and will have a low background. You may also want to try using an EcoRV cut vector instead of a Sma cut vector.

• Remove the oil with Diethylether.
• To 40 ul of the PCR reaction add 50 ul of H2O. Add 10 U T4 PNK, 10 U klenow, ATP (to 1 mM) and some more dNTPs (usually 4 ul of 1.25 mM...whatever) and icubate at 37 oC for 30 min.
• Phenol/chloroform extract
• chloroform/IAA extrac
• EtOH precipitate

you can then just "shotgun" clone this DNA. However, if you have several non-specific bands then you may want to gel purify the fragment first.

One point to note....

We have given up blunt end cloning into Sma I sites where possible. Several people have reported problems with Sma I cut DNA. By choice we clone into EcoRV sites.

X-Gal Protocol (Promega Biotech)

X-Gal Protocol

Caution: X-gal stock solution contains Dimethylformide

X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) turns blue when incubated in the presence of b-glactosidase. This gene is on several of the cloning plasmids (especially, on the pUC series and lGT11 vectors). When an inserted piece of DNA is placed in the correct restriction site, the lacZ gene is interrupted and the colony does not turn the media blue (colony we want). Be sure to run controls.

1. Make 13X100 tubes with 2.5 ml LB and 0.8 agar by melting then dispensing it into the tubes and autoclaving it for only 5 min.
2. Use the tubes while hot or re-melt briefly and hold at 42 C.
3. Add 20 �l 20 mg/ml IPTG (filter sterilized in H2O), 50 �l 20 mg/ml X-gal (in Dimethylformide), and 1 �l Crb solution (100 mg/ml).
   Note: Dimethylformide seems to melt plastic so make the stock in glass or PP (or PA) oakridge tubes.
4. Add the transformed cells (try to get about 200 CFU in up to 250 �l). Vortex and overlay on a CA plate containing the appropriate antibiotic (usually Crb).
   * 2.5 ml is a little tricky to overlay neatly: do not replace cap, tilt the plate to get uniform coverage of the overlay.
5. Let the agar solidify then incubate at 37 C until the blue color develops.
6. Pick the colorless colonies to media with the correct antibiotic and verify the insert by mini plasmid preparations or colony hybridization.

Isolation of Proteus Sp.

Method for isolation and identification of Escherichia coli 0157:H7 and plating media for said process

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Isolation from Lunginfection

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Biotransformation of crotonobetaine to L(-)-carnitine in Proteus sp.

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Method for isolation and identification of Escherichia coli 0157:H7 and plating media for said process

Abstract

Isolation and characterization of phorate degrading soil
bacteria of environmental and agronomic significance

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Impacts Of Antibiotic-resistant Bacteria

By DIANE Publishing
Company

Published 1996
DIANE Publishing

183 pages

ISBN 0788130439

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