yuva ......vibrant: August 2009

Sunday, August 30, 2009

DRUG

Drug Listing: Autonomic Pharmacology

* Direct Muscarinic Agonists
o Choline Esters
o Alkaloids
* Direct Nicotinic Agonist
* Acetylcholinesterase Inhibitor (Reversible)
* Acetylcholinesterase Inhibitor (Irreversible)
* Muscarinic Antagonist
* 2-PAM
* Ganglionic Blockers

* Catecholamines
* Direct Adrenoceptor Agonists
* Indirect-Acting Sympathomimetics
* Alpha adrenoceptor Antagonists
* Beta-adrenoceptor Antagonists
* Adrenergic Neuron Blocking Drugs

Direct Muscarinic Agonists

Choline Esters

* Acetylcholine
* Bethanechol (Urecholine)
* Carbachol
* Methacholine (Provocholine)

Alkaloids

* Muscarine
* Pilocarpine (Pilocar)

Direct Nicotinic Agonist

Nicotine

Acetylcholinesterase Inhibitors

Acetylcholinesterase Inhibitors ("Reversible")

* Neostigmine (Prostigmin)
* Physostigmine (Antilirium)
* Edrophonium (Tensilon)

Acetylcholinesterase Inhibitors ("Irreversible")

* Soman
* Parathion
* Malathion
* Isoflurophate (Floropryl)
* (Diisopropylflurorphosphate DFP)
* Echothiophate (Phospholine)

Muscarinic Antagonists

* Atropine
* Scopolamine
* Ipratropium (Atrovent)
* Pirenzepine (M1 selective)

2-PAM: Acetylcholinesterase Reactivator

* Pralidoxime (Protopam) {2-PAM}:peripheral acetylcholinesterase reactivator for certain phosphoryl-enzyme complexes

Ganglionic Blockers

* Mecamylamine (Inversine)
* Hexamethonium
* Trimethaphan

Catecholamines

DRUG

Epinephrine

Norepinephrine (Levophed)

Isoproterenol (Isuprel)

Dobutamine (Dobutrex)

Dopamine (Intropin)

Receptor Classification

alpha-1, alpha-2, beta-1, beta-2

alpha-1, alpha-2, beta-1

beta-1, beta-2

beta-1 (alpha-1)

D-1 (alpha-1 and beta-1 at high doses)

Direct Adrenoceptor Agonists

Drug

Phenylephrine (Neo-Synephrine)

Methoxamine (Vasoxyl)

Oxymetazoline (Afrin)

Clonidine (Catapres)

Ritodrine (Yutopar)

Terbutaline (Brethine)

Albuterol (Ventolin,Proventil)

Salmeterol (Serevent)

Receptor Classification

alpha-1

alpha-1

alpha-1, alpha-2

alpha-2

beta-2

beta-2

beta-2

beta-2

Indirect-Acting Sympathomimetics

Drug

Ephedrine, Pseudoephedrine

Cocaine

Tyramine

Amphetamine

Mechanism of Action

Release & direct receptor activation

Uptake I inhibitor

Release

similar to ephedrine, but greater CNS actions

Alpha-Adrenoceptor Antagonists

Drug

Prazosin (Minipress)

Terazosin (Hytrin)

Trimazosin

Doxazosin (Cardura)

Phentolamine (Regitine)

Phenoxybenzamine (Dibenzyline)

Tolazoline (Priscoline)

Labetalol (Trandate, Normodyne)

Yohimbine (Yocon)

Receptor Selectivity(alpha1 vs. alpha2)

alpha-1

alpha-1

alpha-1

alpha-1

non-selective

only slightly selective for alpha-1 (non-competitive)

non-selective

alpha-1 (also non-selective beta-antagonist)

alpha-2

ß-Adrenoceptor antagonists

Drug

Propranolol (Inderal)

Metoprolol (Lopressor)

Esmolol (Brevibloc)

Atenolol (Tenormin)

Nadolol (Corgard)

Timolol (Blocadren)

Pindolol (Visken)

Labetalol (Trandate, Normodyne)

Butoxamine

Receptor Selectivity

non-selective

beta-1

beta-1

beta-1

non-selective

non-selective

non-selective (partial agonist)

non-selective (selective alpha-1-antagonist)

beta-2 (no clinical applications)

Adrenergic Neuron Blocking Drugs
Reserpine

Guanethidine (Ismelin)

* non-selective blockade of vesicular uptake and storage of biogenic amines

* similar to reserpine: Uptake I dependent

DRUG LISTING

DOBUTAMINE

More about dobutamine

*

Dobutamine is a racemate; the (+) isomer produces the beta-1 effect (positive-chronotropic), but minimal cardioacceleration occurs because the (-) isomer is an alpha agonist, which tends to prevent the chronotropic effect (cardioacceleration)

*

The result is that cardiac output is increased with minimal increase in heart rate.

Saturday, August 29, 2009

CATACHOLAMINE SYNTHESIS

Catecholamine Synthetic Pathway

Adrenergic Neurotransmission: Introduction to the Neurotransmitters

Norepinephrine: transmitter released at most postganglionic sympathetic terminals

Dopamine: major CNS neurotransmitter of mammalian extrapyramidal system and some mesocortical and mesolimbic neurononal pathways.
Epinephrine: most important hormone of the adrenal medulla
Catecholamine Synthesis, Storage, and Release

Aromatic L-amino acid decarboxylase (DOPA decarboxylase)

dopa leads to dopamine
methyldopa leads to a-methyldopamine (converted by dopamine ß hydroxylase to the "false transmitter" alpha-norepinephrine)
5-hydroxy-L-tryptophanleads to5-hydroxytryptamine (5-HT)
Tyrosine Hydroxylase
tyrosine leads to DOPA
rate limiting step in pathway
tyrosine hydroxylase is a substrate for cAMP-dependent and Ca2+ - calmodulin-sensitive protein kinase and protein kinase C
Increased hydroxylase activity is associated with the phosphorylated enzyme

SITES OF CHOLINERGIC ACTION

Cholinergic Transmission: Site Differences

Skeletal Muscle

Neurotransmitter: Acetylcholine

Receptor Type: Nicotinic

Sectioning and degeneration of motor and post-ganglionic nerve fibers results in:

an enhanced post-synaptic responsiveness, denervation hypersensitivity.

Denervation hypersensivity in skeletal muscle is due to

increased expression of nicotinic cholinergic receptors

and their spread to regions aways from the endplate.

Autonomic Effectors

Neurotransmitter: Acetylcholine

Receptor type: Muscarinic

effector coupled to receptor by a G protein

In smooth muscle and in the cardiac conduction system, intrinsic electrical activity and mechanism activity is present, modifiable by autonomic tone.

Activities include propagated slow waves of depolarization: Examples: intestinal motility and spontaneous depolarizations of cardiac SA nodal pacemakers.

Acetylcholine decreases heart rate by decreases SA nodal pacemaker phase 4 depolarization.

The cardiac action potential associated with HIS-purkinje fibers or ventricular muscle consists of five phases

Phase 0 corresponds to Na+ channel activation.

The maximum upstroke slope of phase 0 is proportional to the sodium current.

Phase 0 slope is related to the conduction velocity in that the more rapid the rate of depolarization the greater the rate of impulse propagation.

Phase 1 corresponds to an early repolarizing K+ current. This current like the Phase 0 sodium current is rapidly inactivated.

Phase 2 is the combination of an inward, depolarizing Ca2+ current balanced by an outward, repolarizing K+ current (delayed rectifier).

Phase 3 is also the combination of Ca2+ and K+ currents.

Phase 3 is repolarizing because the outward (repolarizing) K+ current increases while the inward (depolarizing) Ca2+ current is decreasing.

Phase 4 in normal His-Purkinje and ventricular muscle cells is characterized by a balance between outward Na+ current and inward K+ current. As a result, the membrane potential would normally be flat.

In disease states or for other cell types (SA nodal cells) the membrane potential drifts towards threshold. This phenomenon of spontaneous depolarization is termed automaticity and has an important role in arrthymogenesis.

Rate of phase 4 depolarization is decreased by an increase in K+ conductance--which leads to membrane hyperpolarization (takes longer to reach threshold)

Autonomic Ganglia
Neurotransmitter: Acetylcholine
Receptor type: Nicotinic
Generally similar to skeletal muscle site: initial depolarization is due to receptor activation. The receptor is a ligand-gated channel.
Blood vessels
Choline ester administration results in blood vessel dilatation as a result of effects on prejunctional inhibitory synapses of sympathetic fibers and inhibitory cholinergic (non-innervated receptors).
In isolated blood vessel preparations, acetycholine's vasodilator effects are mediated by activation of muscarinic receptors which cause release of nitric oxide, which produces relaxation.
Signal Transduction

Nicotinic Receptors

Ligand-gated ion channels

Agonist effects blocked by tubocurarine

Receptor activation results in:

rapid increases of Na+ and Ca2+ conductance

deplorization

excitation

Subtypes based on differing subunit composition: Muscle and Neuronal Classification

Muscarinic Receptors

G-protein coupled receptor system

Slower responses

Agonist effects blocked by atropine

At least five receptor subtypes have been described by molecular cloning. Variants have distinct anatomical locations and differing molecular specificities

CHOLINERGIC TRANSMISSION

Cholinergic Neurotransmission

Transmitter Synthesis and Degradation

Acetylcholine is synthesized from the immediate precursors acetyl coenzyme A and choline in a reaction catalyzed by choline acetyltransferase (choline acetylase).

Acetylcholinesterase

Rapid inactivation of acetylcholine is mediated by acetylcholinesterase.

Acetylcholinesterase is present at ganglia, visceral neuroeffector junctions, and neuromuscular junctional endplates.

Another type of cholinesterase, called pseudo-cholinesterase or butyrylcholinesterase has limited presence in neurons, but is present in glia. Most pseudocholinesterase activity is found in plasma and liver.

Pharmacological effects of anti-cholinesterase drugs are due to inhibition of acetylcholinesterase.

Acetylcholine Storage and Release

Small random release of acetylcholine-quanta, producing miniature end-plate potentials (mepps) , are released by presynaptic terminals.
These small currents were linked to ACh release since anticholinesterases (neostigmine) increased their effects, while cholinergic receptor antagonist (tubocurarine, a nicotinic receptor blocker) blocked.
Anatomical counterpart to the electrophysiological quanta is the synaptic vesicle.
The model is based on the nicotinic, skeletal neuromusclar junction.
Synchronous exocytotic release of many more quanta, dependent on Ca2+ occur when an action potential reaches the terminal.
Exocytotic release of acetylcholine and other neurotransmitters is inhibited by toxins elaborated by Clostridium botulinum.
Botulism

Botulism is caused by the most potent neurotoxins known. The neurotoxins are produced and liberated by Clostridium botulinum.
C. botulinum, ubiquitously found in soil and marine environments, is a group of gram positive anerobes that form spores.

Eight distinct toxins have been characterized, all but one being neurotoxic.
Botulinum neurotoxin affects cholinergic nerve terminals:
postganglionic parasympathetic endings
neuromuscular junctions
peripheral ganglia
CNS is not involved.
Botulinum neurotoxin prevents acetylcholine release:
binds presynaptically
internalized in vesicular form
released into the cytoplasm
the toxin(s), (zinc endopeptidases) causes proteolysis of components of the neuroexocytosis system.

function of ans

Fight or Flight: General Functions of the Autonomic Nervous System

ANS regulates organs/processes not under conscious control including:

circulation

digestion

respiration

temperature

sweating

metabolism

some endocrine gland secretions

Sympathetic system is most active when the body needs to react to changes in the internal or external environment: The requirement for sympathetic activity is most critical for:

temperature regulation

regulation of glucose levels

rapid vascular response to hemorrhage

reacting to oxygen deficiency

During rage or fright the sympathetic system can discharge as a unit--affecting multiorgan systems.

Sympathetic fibers show greater ramification.

Sympathetic preganglionic fibers may traverse through many ganglia before terminiating at its post-ganglionic cell. Synaptic terminal arborization results in a single preganglionic fiber terminating on many post-ganglionic cells.

This anatomical characteristic is the basis for the diffuse nature of sympathic response in the human and other species.

Sympathetic Responses

heart rate increases

blood pressure increases

blood is shunted to skeletal muscles

blood glucose increase

bronchioles dilate

pupils dilate

Parasympathetic responses

slows heart rate

protects retina from excessive light

lowers blood pressure

empties the bowel and bladder

increases gastrointestinal motility

promotes absorption of nutrients

parasympathetic nervous system

Comparisons between Sympathetic and Parasympathetic Nerves

Sympathetic system has a broader distribution, innervating effectors throughout the body

Sympathetic fibers show greater ramification.

Sympathetic preganglionic fibers may traverse through many ganglia before terminiating at its post-ganglionic cell.
Synaptic terminal arborization results in a single preganglionic fiber terminating on many post-ganglionic cells.
This anatomical characteristic is the basis for the diffuse nature of sympathic response in the human and other species.

Parasympathetic system is relatively limited

The parasympathetic system has its terminal ganglia near the end-organ.

Sometimes there is but a one-to-one ratio relationship between pre-and post-ganglionic fibers. The ratio between preganglionic vagal fibers and ganglion cells may be much higher, e.g. 1:8000 for Auerbach's plexus

reflex arc

Autonomic Reflex Arc

First link: Visceral autonomic afferents to the CNS
Non-myelinated, carried to the cerebrospinal axis by autonomic nerves (e.g.vagus and splanchnic)
Some autonomic afferents from skeletal muscle blood vessels and integumental structures may be carried in somatic nerves
Cell bodies of visceral afferents: (a) spinal nerves--in dorsal root ganglia; (b) cranial nerves-- in sensory ganglia
What information gets transmitted?
Mediated Information:
visceral sensation (pain;referred pain)
vasomotor reflexes
respiratory reflexes
viscerosomatic reflexes: Definition: Viscerosomatic: Pertaining to the viscera and body .

autonomic nervous system

Autonomic and Somatic Innervation

Skeletal muscle is innervated by somatic nerves, controlling voluntary actions
All other innervated structures are supplied by the autonomic or involuntary system.
Somatic system: No ganglia present
Autonomic nervous system (ANS) has ganglia.
these ganglia are sites at which preganglionic fibers form synaptic connections with postganglionic neurons
these ganglia are located outside the cerebrospinal axis
Other differences between Somatic and Autonomic Innervation
Motor nerves to skeletal muscle: myelinated
Postganglionic autonomic nerves are nonmyelinated
Denervation of skeletal muscle results in paralysis and atrophy
Denervated smooth muscle or glands retain some activity .

Wednesday, August 26, 2009

gel electrophoresis chamber

ELECTROPHORESIS CHAMBER

Constructing your own Plexiglas® electrophoresis chamber is quite easy, inexpensive and fun. After you have made your chamber, try it out using the Colorful Electrophoresis experiments.

SAFETY: Remember to wear safety glasses at all times while constructing this chamber.

TIP: Leave the paper on each Plexiglas piece until you are ready to use it. Write the name of each piece of Plexiglas® on the paper covering the surface before beginning construction. Remove the paper as you work with each piece.

Bonding Plexiglas® pieces together

Bonding pieces of Plexiglas together is most easily accomplished with two people working together. One person holds the pieces in position, and the other person uses a syringe or Pasteur pipette to apply a thin line of acrylic bonding agent along both sides of the seam where the two pieces of Plexiglas meet. The liquid should wick between the two pieces of Plexiglas at the seam and bond them together within 2 minutes (hold them firmly together until bonded). It is very important to hold the pieces together firmly while bonding so that a leak-proof seal is made.

If a mistake is made in gluing pieces together and you discover it before the bond has fully set, the pieces can be snapped apart and re-bonded.
gel carrier

Pieces will bond best if you hold them so that the seam is horizontal. It is also better to hold the pieces so that the seam is not close to the table/surface you are working on. This allows any excess liquid to evaporate quickly. If the seam is close to or on a table/surface, the liquid may wick underneath and smear the Plexiglas.

Step 6
A. Construct the Gel Carrier

1. You will need the following pieces of Plexiglas:
* Gel Carrier Base - 3-3/4" by 3-3/4" by 3/4" thick
* Gel Carrier Sides (2) - 3-3/4" by 1/2" by 1/2" thick
2. Cut a slot 3/16" from one end of each Gel Carrier Side. The slots should be 1/16" wide x 1/8" deep; the Teflon® should fit easily into the slots. If desired, cut a second slot 1-5/8" from the first slot. gel carrier side view
3. Remove the paper from the Gel Carrier Sides and the Gel Carrier Base.
4. Lay the Gel Carrier Base on a flat surface. Lay the Gel Carrier Sides on top, on opposite edges, along the sides.
TIP: Make sure that the slots in the Gel Carrier Sides line up.
5. Turn the Gel Carrier Base on its edge, and bond the top Gel Carrier Side to it. Turning the Gel Carrier Base on its edge while bonding prevents the bonding agent from flowing underneath and smearing the Plexiglas.
6. Turn the Gel Carrier Base on the other edge, and bond the other Gel Carrier Side to it.

Step 5

Step 8
B. Cut the Gel Comb

1. You will need the 3-5/8" by 1" by 1/16" thick Teflon piece.
2. Copy the comb pattern onto paper or paperboard and cut it out.
3. Place the pattern in the gel carrier slots and check to make sure that the bottom edges of the comb teeth will be at least 1/16" (2 mm) above the Gel Carrier Base. It is very important that the wells in the gel formed by the teeth not go all of the way through the gel. Modify the pattern if needed.
4. Use a permanent marker to trace the pattern on the Teflon or tape the pattern down on the Teflon.
5. Place the Teflon on a piece of scrap lumber or cardboard and cut out the comb using an X-acto (TM) knife.
6. Use fine sandpaper (150 grit) to smooth the edges of the Comb teeth so that they will not rip the wells in the gel when the Comb is removed.
7. Place the comb in the slots in the Gel Carrier sides. If it does not slide in and out of the slots easily, sand the flat surface of the Teflon so that it does slide easily.
8. Again place the comb in the slots in the Gel Carrier sides. Check that the bottom edge of each comb tooth is at least 2mm (1/16") above the Gel Carrier Base. If any are not, sand them down.

comb pattern (click on image for printing instructions)
Step 7

Step 7

Step 10
C. Drill holes for the leads to the power supply

1. You will need the following pieces of Plexiglas:
* Chamber Long Side - one of the 7-1/2" by 2-1/2" by 1/4" thick pieces
* Chamber Lid Front - 7-1/2" by 1-1/4" by 1/4" thick

TIP: Do not remove the paper from these pieces until you are ready to bond them together

drilling holes
2. Lay the Chamber Lid Front on top of the Chamber Long Side, matching the two pieces along one long edge and the two sides. Tape the two sides together with masking tape so that the pieces do not slip while drilling.
3. Mark "X's" on the top edges and "X's" on the paper of the sides facing you. This will assist you in bonding the pieces together in the correct orientation later.
4. On the Chamber Lid Front, measure in 1" from each short end and make a pencil mark. Measure down 5/8" from one long edge and make cross marks in the centers of the first marks; you should have a "+" mark at each end of the Lid Front.
5. Place the point of a nail at the center of each "+" mark and tap the head of the nail with a hammer to make a guide hole for the drill bit.
6. If you are using an electric hand drill, place the Plexiglas pieces on top of a piece of scrap lumber. Place a C-clamp in the center of the long edges of the Plexiglas and clamp the Plexiglas/wood sandwich onto a table top in preparation for drilling.
7. Use a 9/64" drill bit to drill holes through both pieces of Plexiglas® in the places you made the guide holes (in the center of the "+" marks).
TIP: Use a small brush to lightly oil the bit before drilling. This will help keep the Plexiglas® from bonding to the bit as it heats up during drilling.
8. Unclamp the Plexiglas and remove the masking tape holding the two pieces together.

Chamber Lid Front
9. Clamp the Chamber Lid Front down for drilling with the side opposite the "X" up. Remember the piece of scrap lumber if you are using an electric hand drill.
10. Use the 5/16" bit to drill through the entire thickness of the Lid Front, centering the tip of the drill in the 9/64" holes
TIP: Remember to lightly oil the bit before drilling.
11. Unclamp the Lid Front.

Step 4
D. Construct the Chamber Lid

1. You will need the following pieces of Plexiglas:
* Chamber Lid Front - 7-1/2" by 1-1/4" by 1/4" thick piece with holes drilled in it
* Chamber Lid - one of the 7-1/2" by 5" by 1/4" pieces

Chamber Lid
2. Remove only the paper marked with an "X" from the Chamber Lid Front. Use a marker to place an "X" on the Plexiglas®. Now remove the paper from the other side.
3. Remove the paper from the Chamber Lid.
4. Bond the Chamber Lid Front to the Chamber Lid. The "X" on the face of the Chamber Lid Front should face outward; the "X" on the edge should be against the Chamber Lid.

Step 6a

Step 6b

Step 6c
E. Construct the Electrophoresis Chamber

1. You will need the following pieces of Plexiglas:
* Chamber Bottom - 7-1/2" by 5" by 1/4" thick
* Chamber Long Sides (2) - 7-1/2" by 2-1/2" by 1/4" thick, one of which has holes drilled in it
* Chamber Short Sides (2) - 3-7/8" by 2-1/2" by 1/4" thick

Electrophoresis Chamber
2. Remove the paper from the Chamber Bottom and lay it on a flat surface.
3. Remove only the paper marked with an "X" from the Chamber Long Side that has holes drilled in it. Use a marker to place an "X" on the Plexiglas®. Now remove the paper from the other side.
4. Remove the paper from the other pieces of Plexiglas®.
TIP: The following bonding steps require two people working together and/or the use of large rubber bands.
5. Use the Chamber Long Sides and the Chamber Short Sides to form a box on top of the Chamber Bottom; the 2-1/2" sides are perpendicular to the base. The Chamber Short Sides should be on the insides of the Chamber Long Sides. Hold the pieces in place, making sure that there are no gaps. The Chamber Long Side that has "X's" marked on it should be oriented as follows: the X on the surface should be on the outside; the X on the edge should be on the top.
TIP: Slightly inset the Chamber Short Sides from the edge of the base. This will keep the bonding agent from flowing under the Chamber Bottom and smearing it.
TIP: Use two large rubber bands to hold the sides in position while you are bonding them.
6. First bond the bottom edges of the square to the Chamber Bottom. Then turn the chamber on successive sides so that the seams you are bonding are horizontal. Remember to place bonding liquid on both the inside and outside edges of each seam.
7. Fill the chamber with water to test for leaks. Re-seal seams with the bonding agent if you find any leaks.

Step 4

Step 5
F. Place the pegs in the Electrophoresis Chamber

1. You will need the following pieces of Plexiglas®:
* Electrophoresis Chamber (from previous step)
* Gel Carrier (already constructed)
* 6 Pegs - 1/2" long pieces of 3/8" dowel or rod

Peg Placement
2. Sand the bottoms of the pegs to remove any burrs; they must be smooth to bond well.
3. Place the Gel Carrier in the center of the Electrophoresis Chamber with the Gel Carrier Sides parallel to the long sides of the Chamber.
4. Bond a peg on the bottom of the Chamber on either side of the Carrier; the pegs are centered along the two edges of the Carrier and are not tight against it. Remove the Gel Carrier from the Electrophoresis Chamber.
TIP: To bond the pegs, grasp one end with forceps/tweezers. Dip the other end into the acrylic bonding agent and then place the peg in the chamber. Remember to turn the chamber so that the surface on which you are placing the peg is horizontal.
5. Place the chamber on one short side. Bond 2 pegs on this side, approximately 3/8" up from the bottom of the chamber and 1/2" in from the sides.
6. Place the chamber on its other short side and repeat the process.

Steps 2-5

Step 4a

Step 4b

Steps 6-8

Steps 9-10

Steps 11-13

Completed box
G. Wire the Electrophoresis Chamber

1. You will need:
* 2 Insulated Banana Jacks
* 2 Solderless Banana Plugs
* 2 nuts to fit Solderless Banana Plugs
* 2 Stackable Banana Plugs
* 2 feet each of black and red Test Lead Wire
* Monel Seizing Wire or 20-gauge copper wire - 16" long piece cut in 1/2
* 2 - 1" pieces of 1/4" diameter heat shrinking tubing, one black and one red

Chamber Wiring
2. Install a Solderless Banana Plug in each of the two holes you drilled in the Electrophoresis Chamber Long Side. The Plug ends should face outward, with a nut on the inside of the chamber. Use a small adjustable wrench to help tighten the Plugs in place.
3. Place the second nut on the threaded side of the Plug but do not tighten it.
4. Use one piece of Monel or copper wire to form the electrode on the right side of the Electrophoresis Chamber. Form a small loop in one end of the wire. Place the loop between the two nuts on the Solderless Banana Plug. Tighten the nuts on the wire to form a good electrical connection. Loop the wire once around the peg closest to the Jack, then loop it around the other peg. Cut off any excess wire. Do not solder this wire into place since you will need to change it when it wears out. If you use copper wire, it will react with the electrode buffer, turning the buffer blue. Use new buffer for each gel run.
5. Repeat Step 4 on the left side of the Electrophoresis Chamber.

Chamber Wiring Banaplugs
6. Install the Banana Jacks in each of the two holes you drilled in the Electrophoresis Chamber Lid Front. Place the black Jack in the right hole and the red Jack in the left hole; the terminal ends and nuts face outward. Use a wrench to tighten the nuts.
7. Use wire strippers to strip off about 3/4" of insulation from the end of the black Test Lead Wire.
8. Thread the stripped Wire end through the hole in the metal part of the Jack and twist the stripped wire back upon itself. Make sure it feels tight.
9. Thread the piece of black heat shrinking tubing over the Test Lead Wire. Make sure to thread over the metal piece on the back of the Jack. Use an alcohol lamp or hair dryer (high heat setting) to heat the tubing to shrink it around the pieces. Do not place the tubing directly in the flame. Rotate the pieces in the heat to shrink the tubing evenly.
10. Repeat Steps 7 - 9 with the red Banana Jack, red Wire, and red heat shrinking tubing.
11. Strip about 1/2" of insulation off of the end of the black Wire (there is a Banana Jack at the other end).
12. Take a small screwdriver and unscrew the screw inside the black Stackable Banana Plug. Do not completely remove the screw. Double the stripped end of Wire on itself and thread it through the opening of the Plug. Then tighten the screw to keep the Wire in place.
13. Repeat Steps 11-12 with the red Wire and red Stackable Banana Plug.
14. Optional: Place a thin strip of black electrical tape across the bottom of the Gel Carrier under the comb. The black surface makes it easier to visualize the wells when loading the gel. Note: The black surface will make it difficult to visualize food color or dye separation in the black area; so place the electrical tape so that it does not extend past the wells toward the long end of the gel.

yuva ......vibrant