Ibi QS-710 Manuale utente

IBI SCIENTIFIC

QS-710 - QUICK SCREEN

HORIZONTAL GEL ELECTROPHORESIS UNIT

GEL SIZE:7CM X 10CM

IBI Catalog Number: IB51000

OPERATOR’SMANUAL

SCIENTIFIC

TABLE OF CONTENTS

A. SAFETY INFORMATION 3

B. PACKAGE CONTENTS 3

C. PRODUCT SPECIFICATIONS 3

D. OPERATING INSTRUCTIONS 4

PREPARATION OF THE AGAROSE GEL - DNA 4

PREPARATION OF THE AGAROSE GEL - RNA 5

CASTING THE GEL 6

REMOVING THE COMB 7

LOADING THE SAMPLES INTO THE GEL 7

ELECTRICAL CONNECTIONS TO THE SAFETY LID 8

SAMPLE ELECTROPHORESIS 8

DETECTION AND DOCUMENTATION OF SEPARATED FRAGMENTS 9

CHOICE OF BUFFER 9

VOLTAGE 10

STAINING SOLUTION 10

E. MAINTENANCE OF UNIT 11

F. REPLACEMENT PARTS & ACCESSORIES 11

QS-710 ACCESSORY ITEMS AND REPLACEMENT PARTS 11

QS-710 COMBS 12

G. RELATED IBI PRODUCTS 12

H. RELATED IBI CERTIFIED REAGENTS 12-14

I. REFERENCES 15

J. LIMITED WARRANTY 15

A. SAFETY INFORMATION

Important Safety Information!

Please read this manual carefully before operating your new IBI QS-710 unit.

This manual contains important operating and safety information.

To best use the product, please read the entire manual carefully prior to use.

To avoid possible injury, this product should only be used for its intended purpose.

B. PACKAGE CONTENTS

Upon receiving this product, please verify all of the noted parts and accessories are contained in

this package.

Model QS-710 Buffer Tank

Patented QS-710 Vented Lid

UV Transmittant Casting Tray

Casting Fixture

One Set of 1.5mm x 8 Tooth Combs (2 ea.)

One Set of Power Cords (Red and Black)

Bubble Level

Operation Manual

NOTE: Carefully inspect all items in the package to insure no items are broken or missing. If

there are items broken, please inspect the package carefully for signs of shipping damage.

If there is ANY sign of shipping damage, please contact the carrier and file a claim with

them immediately. Contact the distributor from which you purchased the item.

C. PRODUCT SPECIFICATIONS

Height Width Length

Unit Dimensions 6.4cm 11.4cm 22.2cm

Gel Dimensions 7.0cm 10.0cm

Maximum Sample Capacity: 30 Samples - 2 Combs, 15 samples each

Buffer Capacity: 250ml

Distance Between Electrodes: 19.3cm

D. OPERATING INSTRUCTIONS

Your new QS-710 Quick Screen Horizontal Unit is cleaned and wiped prior to packaging; however,

components should be washed in warm soapy water prior to use in the laboratory. A mild dish wash-

ing liquid, like Joy, works well.

Gently wash the tank, lid, UVT casting tray, and casting fixture in warm soapy water, taking care not

to scratch any of the acrylic components such as the tank and UVT tray. Do NOT wash Power Cords.

NOTE: It is also recommended that the UVT casting tray be cleaned with alcohol prior to use. Be

certain the entire unit is dry prior to use.

PREPARATION OF THE AGAROSE GEL - DNA

1.) Select the percentage gel necessary to effectively resolve your sample, use Table 1 as a guide.

Table 1 Gel Concentrations and Resolving Ranges

Concentration of Efficient Range of

Agarose in Gel Separation of Linear DNA

(% w/V) (Kb)

0.3% 5 - 60

0.6% 1 - 20

0.7% 0.8 - 10

0.9% 0.5 - 7

1.2% 0.4 - 6

1.5% 0.2 - 3

2.0% 0.1 - 2

* Table taken from Sambrook, J., Fritsch, E.F., & Maniatis, T. (1989)

Molecular Cloning, A Laboratory Manual, 1, 6. 8, 613.

2.) Weigh an appropriate quantity of agarose (0.3% means 0.3gm of agarose per 100ml of gel

volume) and place it into a 250ml flask.

3.) Make up 500ml of either 1X TAE or 1X TBE electrophoresis buffer. See below:

Electrophoresis Buffers

The two most commonly used buffers for horizontal electrophoresis of double stranded DNA in

agarose gels are Tris-Acetate-EDTA (TAE) [IB70160] and Tris-Borate-EDTA (TBE) [IB70150].

While the resolving powers of these buffers are very similar, the relative buffer capacities are very

different, conferring different run attributes which are summarized below:

TAE (IB70160): Tris-acetate has traditionally been the more commonly used buffer. However, its

relatively low buffer capacity will become exhausted during extended elec-

trophoresis, making buffer recirculation necessary in runs exceeding 140 mA-

hours. Potential advantages of using TAE buffer over TBE buffer include superi-

or resolution of supercoiled DNA and approximately 10 % faster migration of

double-stranded linear DNA fragments.

TBE (IB70150): Tris-borate's significantly greater buffering capacity and its relatively low current

draw eliminates the need for recirculation in all but the most extended runs (>

300 mA-hours). TBE buffer systems are not recommended when fragments are to

be recovered from the gel after electrophoresis.

4.) Add ethidium bromide (IB40075) to the diluted electrophoresis buffer to a final concentration

of 0.5μg/ml.

NOTE: The addition of ethidium bromide to both the gel and the running buffer will result in

maximum detection levels by providing high levels of sample fluorescence with an evenly

low level of background.

5.) Add 6.6ml of the 1X electrophoresis buffer containing ethidium bromide made in step 4 per

millimeter of gel thickness desired, up to a maximum of 50ml, to the flask containing the

agarose (IB70035-40-42-45). A 50ml gel solution will make a 7.6mm thick gel. Thinner gels

may be made, however care must be taken that the wells are deep enough to accommodate the

desired sample volume.

Sample Volume

Catalog # Comb Description Well Width Per mm Gel

IB51040 1.0mm, 8 tooth 4.7mm 4.7ul

IB51045 1.0mm, 12 tooth 2.8mm 2.7ul

IB51050 1.0mm, 15 tooth 2.2mm 2.3ul

IB51055 1.5mm, 8 tooth 4.7mm 7.0ul

IB51060 1.5mm, 12 tooth 2.8mm 4.0ul

IB51065 1.5mm, 15 tooth 2.2mm 3.3ul

IB51070 1.0mm, 0 Marker, 1 Sample 62.6mm 62.6ul

IB51075 1.0mm, 1 Marker, 1 Sample 4.7mm, 54.7mm 4.7ul, 54.7ul

IB51080 1.5mm, 0 Marker, 1 Sample 62.6mm 94ul

IB51085 1.5mm, 1 Marker, 1 Sample 4.7mm, 54.7mm 7.0ul, 82ul

6.) Make note of the total solution volume so that a degree of evaporation can be determined and

corrected for.

7.) Heat the agarose slurry in a microwave oven for 90 seconds. Swirl the flask to make sure any

grains sticking to the walls enter into the solution, undissolved agarose appears as small

"lenses" floating in the solution. Heat the solution for an additional 30-60 seconds.

Re-examine the solution and repeat the heating process until the agarose completely dissolves.

8.) Add deionized water to replace any volume lost through evaporation during the heating process.

Proceed to "Casting the Gel" on page 6.

PREPARATION OF THE AGAROSE GEL - RNA

RNA molecules are separated by electrophoresis through denaturing gels prior to analysis by northern

hybridization. Agarose gels containing formaldehyde are commonly used for RNA electrophoresis.

Presented below is a general protocol for electrophoresis of RNA using formaldehyde gels.

CAUTION! All equipment and solutions used in the following protocol should be treated with DEPC

(diethyl pyrocarbonate) or acetic anhydride prior to use to inhibit RNase activity. It is

recommended that dedicated solutions be made solely for RNA work to minimize the risk

of sample degradation due to RNase activity.

NOTE: Staining RNA samples with ethidium bromide has been reported to reduce sample blotting

efficiency. Therefore, if samples are to be analyzed by northern hybridization after elec-

trophoresis, run a duplicate lane(s) for staining, or minimize the exposure of RNA samples

to ethidium bromide by following the post-electrophoresis staining protocol on page 10.

The following protocol will make 50ml of a 1.5% agarose gel containing 1X MOPS [3-(N-

Morpholino)-Propanesulfonic Acid]-Acetate-EDTA (MAE) buffer and 2.2M formaldehyde, result-

ing in a 7.5mm thick gel:

1.) Weigh out 0.5gm of agarose, and place into a 125ml flask.

2.) Add 43.5ml of DEPC (or acetic anhydride) treated water.

3.) Make note of the total solution volume so that degree of evaporation can be determined and

corrected for.

4.) Heat the agarose slurry in a microwave oven for 60 seconds. Swirl the flask to make sure any

grains sticking to the walls enter into the solution. Undissolved agarose appears as small

"lenses" floating in the solution. Heat for an additional 30-60 seconds. Re-examine the solution

and repeat the heating process until the agarose completely dissolves.

5.) Add deionized water to replace any volume lost through evaporation during the heating process.

6.) Allow the solution to cool to 60oC. Place the flask in a hood and add 5ml of 10X MAE buffer,

and 1.5ml of 37% formaldehyde.

CAUTION: Formaldehyde vapors are toxic. Gel preparation should take place in a hood and solu-

tions and gels containing formaldehyde should be kept covered when possible.

CASTING THE GEL

1.) Place the gel casting fixture on a lab bench. Check

to see that it is level by placing the supplied level-

ing bubble in the center of the platform. The bub-

ble should appear within the center circle.

CAUTION! Cast agarose gels containing

formaldehyde in a hood.

2.) Insert the gel casting tray into the casting fix-

ture by pressing one end of the tray against the

thick foam backed casting pad. While com-

pressing the foam pad with the casting tray,

move the other end of the tray down into the

casting fixture (see Photo 1).

3.) When the gel solution has cooled to approxi-

mately 55oC, slowly pour it into the gel tray. If

hotter gel solutions are routinely poured, the

tray may warp over time.

4.) If bubbles form on the surface of the gel upon

pouring, use the comb to either pop them or lightly

brush them to the sides of the gel. If large bubbles

are allowed to harden within the gel, they may

cause artifacts to occur during electrophoresis.

5.) Insert one or more combs by placing them into the

slots in the casting tray. For best results, place the

comb in the slot nearest the thin pad of the casting

fixture. If two combs are desired, place the second

in the center comb slot (see Photo 2).

6.) Allow the gel to harden undisturbed for at least

30 minutes.

Photo 1

Photo 2

REMOVING THE COMB

1.) When the gel is solidified and fully opaque, carefully remove the comb with a gentle wiggling,

upward motion. If the comb is difficult to remove or if a low percentage gel is being used,

overlay the comb area with a small volume of 1X electrophoresis buffer to preserve the integri-

ty of the wells. Check the wells to ensure their bases are intact.

CAUTION: Prolonged exposure of the Delrin combs to gels containing formaldehyde will cause

them to degrade. Be sure to remove the comb(s) from formaldehyde gels as soon as

gel hardening is complete and rinse them well prior to storage.

If a gel is not to be used immediately after preparation, remove it from the casting fixture and place

it in a plastic bag or container submerged in 1X electrophoresis buffer containing 1mMNaN3. Store

at +4oC.

LOADING THE SAMPLES INTO THE GEL

1.) Remove the casting tray containing the hard-

ened agarose gel from the casting fixture by

pressing the casting tray against the foam pad

and lifting at the thin pad end. Place the tray

and gel into the main unit assembly such that

the samples wells are on the same end as the

negative (black) electrode. (see Photo 3)

2.) Fill the unit with the remaining 1X elec-

trophoresis buffer containing ethidium bro-

mide made previously (or 1X MAE buffer

for RNA gels), covering the gel to a depth of

1-5mm. Approximately 250ml of buffer will

be required.

NOTE: Use of the same batch of electrophoresis

buffer for both the gel and the running

buffer is very important. Slight variations in buffer composition between gel and running

buffer may result in ionic or pH gradients that can significantly impact the mobility of

the samples.

3.) Pre-run RNA gels at 100V for five minutes prior to loading the samples.

4.) Load the samples into the wells with a micropipette or similar device taking care not to punc-

ture the bottom of the wells or load the sample onto the top of the gel. For improved well visu-

alization during sample loading, be sure that the wells are positioned over the contrasting

stripes located on the bottom of the buffer tanks.

Photo 3

ELECTRICAL CONNECTIONS TO THE SAFETY LID

CAUTION: This unit is intended to be used with a power supply which detects a no current con-

dition and prevents a current flow unless there is a completed circuit path. Use of

other power supplies may compromise the safety of this unit.

The QS-710 can only be operated with the safety lid in place. Electrical current is supplied through

the banana plugs to the lid which, in turn, connect to the gold plated tank connectors. A simple

gravity connector in the cover ensures a complete current path, yet allows the lid to be removed

from the unit without disturbing the loaded samples.

1.) Plug the female ends of the black (-) and red

(+) leads into the banana jacks on the rear of

the lid. Be certain that the black (-) plug is

connected to the cathode end, where the sam-

ples originate (see Photo 4).

2.) After the samples have been loaded into the gel,

place the lid over the unit so that the connector

covers align with the round tank connectors.

3.) Set the lid straight down so that the lid rests

squarely on the unit and the round tank connec-

tors are inside the connector covers.

4.) Make sure the power supply is turned off. Plug

the male ends of the red and black leads into a

corresponding set of jacks on the power supply

(black - black; red - red).

CAUTION: Do not jar or bump the gel box once the lid is place. The electrical connection is

made by gravity once the lid is in position. While this design helps to minimize sam-

ple disturbance during lid placement, it also may result in a disruption of power to

the unit if the lid or unit are disturbed during the run.

SAMPLE ELECTROPHORESIS

1.) The QS-710 is designed for quick screen electrophoresis. The maximum suggested applied

voltage for the electrophoresis of DNA in agarose gels using the QS-710 is 100V. In a 1% TBE

gel, this translates into a run time of approximately 1/2 hour. Lower voltages may be used, of

course, and as a general rule, a 50V run will take twice as long as a 100V run. Higher voltages

may be used to decrease run time, however, if the unit is being operated at higher voltages than

100V, the heat generated during electrophoresis may decrease sample resolution. Such artifacts

may be avoided by running the unit in a cold room or adding 1X electrophoresis buffer "ice

cubes" to keep the unit properly cooled.

CAUTION: DO NOT EXCEED THE MAXIMUM OPERATING VOLTAGE OF 175 VOLTS.

The suggested run parameters for the electrophoresis of RNA in agarose gels containing formalde-

hyde is 60-80V. Since the QS-710 has no capacity for buffer recirculation, the buffer from each

reservoir should be collected after 1.5 hours of electrophoresis and properly discarded.

CAUTION: Formaldehyde vapors are toxic. Electrophoresis of RNA in gels containing formalde-

hyde should take place within a fume hood.

Photo 4

2.) Follow the sample migration into the gel using the loading dye as an indicator. (See “Choice of

Buffer” for the Sample Loading Buffer recipe) Allow the samples to migrate until the fragments

have separated, normally until the bromophenol blue dye front has migrated 3/4 of the way down

the gel.

NOTE: If the gel contains ethidium bromide, the progress of electrophoresis may be monitored

during the run by turning off the power supply, removing the lid, and shining a medium-

wave UV light onto the gel. The resolved bands will appear as orange bands against a

dark purple background.

DETECTION AND DOCUMENTATION OF SEPARATED FRAGMENTS

1.) At the completion of the run, turn off the power supply and disconnect the leads. Remove the

lid (it is not necessary to remove the power leads from the lid) and remove the gel tray.

2.) To stain RNA gels containing formaldehyde post electrophoresis, soak the gel in 1L of DEPC-

treated water overnight at room temperature. Transfer the gel to a solution of 20X SSC contain-

ing 0.5μg/ml of ethidium bromide, stain for 5 -10 minutes.

3.) Ethidium bromide stained samples are visualized by exposing them to medium wavelength

(312nm) UV light. Because the gel casting tray is UV transmittant, the gel does not need to be

removed from the tray before viewing. Place the gel casting tray containing the gel on the filter

surface of a UV transilluminator for convenient viewing.

4.) Sample banding patterns may be documented by autoradiography.

CHOICE OF BUFFER

NOTE: Tris-borate buffer allows faster sample migration than tris-acetate buffers with no apparent

loss of resolution. A tris-borate buffer is usually the choice for quick-screening. Phosphate

buffers are used with glyoxal or formaldehyde gels.

The two most commonly used buffers for horizontal electrophoresis of double stranded DNA in

agarose gels are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE). While the resolving

powers of these buffers are vary similar, the relative buffer capacities are very different, conferring

different run attributes which are summarized below:

Tris-Acetate has traditionally been the more commonly used buffer. However, its relativity low

buffer capacity will become exhausted during extended electrophoresis, making buffer recircula-

tion necessary in rums exceeding 140mA-hours. Potential advantages of using TAE buffer over

TBE buffer include superior resolution of supercoiled DNA and approximately 10% faster migra-

tion of the double-stranded linear DNA fragments.

Tris-Borate has a significantly greater buffering capacity and its relativity low current draw elimi-

nates the need for recirculation in all but the most extended runs (>300mA-hours). TBE buffer sys-

tems are not recommended when fragments are to be recovered from the gel after electrophoresis.

TRIS ACETATE EDTA BUFFER (TAE) - IB70160:

1X Working Concentration: 10X Stock Solution:

40 mM Tris base 48.4 g Tris Base

20 mM Glacial Acetic Acid (NaOAc) 16.4 g or 11.42ml NaOAc

2.0 mM EDTA 7.4 g EDTA or 20ml 0.5M EDTA (pH 8.0)

pH 8.3 H2O to 1L

TRIS BORATE EDTA BUFFER (TBE) - IB70150:

1X Working Concentration: 10X Stock Solution:

89 mM Tris Base 108g Tris Base

89 mM Boric Acid 55g Boric Acid

2.0 mM EDTA 6.72g EDTA or 40ml 0.5M EDTA (pH 8.0)

pH 8.0 H2O to 1 liter

MOPS ACETATE EDTA (MAE) - IB70175:

Solutions containing MOPS should be wrapped in aluminum foil and stored at room temperature.

The buffer tends to yellow with age. Light yellow buffer may be used, however, dark yellow solu-

tions should be discarded.

1X Working Concentration: 10X Stock Solution:

20 mM MOPS (pH 7.0) 41.8g MOPS

8 mM NaOAc 800 ml DEPC treated H2O

1 mM EDTA (pH 8.0) adjust pH to 7.0 with NAOH and add:

16.6ml 3M DEPC-treated NaOAc

20.0ml 0.5 M DEPC-treated EDTA, pH 8.0

bring to 1.0 liter and filter

Solutions containing MOPS should be wrapped in aluminum foil and stored at room temperature.

The buffer tends to yellow with age. Light yellow buffer may be used, however, dark yellow solu-

tions should be discarded.

Sample Loading Buffer - DNA Sample Loading Buffer - RNA

10X Stock Solution: 5X Stock Solution:

50 % Glycerol 1 mM EDTA, pH 8.0

100mM Na3EDTA 0.25 % Bromophenol Blue

1% SDS 0.25 % Xylene Cyanol

0.1% Bromophenol Blue 50 % Glycerol

pH 8.0

VOLTAGE

The QS-710 is designed for rapid electrophoresis with moderate resolution. Suggested voltage is

100V for a 30 to 60 minute run. Higher voltages may be used to decrease run time, however, the

volt-hours should remain constant. One should not exceed 200 volt-hours without changing the

buffer in the unit. If the unit begins to operate at voltages higher than 150V we recommend adding

1X electrophoresis buffer ice cubes to keep the unit properly cooled.

STAINING SOLUTION

The simplest staining procedure is to add 1-5g Ethidium Bromide per ml of gel solution just prior

to casting the gel. Alternatively, the gel can be stained in a solution of 5uG ethidium bromide/ml of

1X gel buffer for 15 minutes. Destain in deionized water or 1mM MgSO4for two minutes.

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