Hoefer SE 400 Скачать руководство пользователя страница 39 | Manualshive

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Hoefer SE 400 Скачать руководство пользователя страница 39

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p35

Note:

Some bleaching

may occur if using Destain
solution II as a stop solution.

Soak the gel in 500 ml of deionized water overnight.
The next day, rinse the gel with several changes of
deionized water over 30–60 minutes.
D. Place the gel in 100–200 ml of 5 µg/ml DTT in

deionized water for 30 minutes.

E. Pour off the DTT solution but do not rinse the gel.

Add 100 ml of silver nitrate solution directly to the
gel. Shake gently for 30 minutes and then rinse
the gel for 1–2 seconds with deionized water.

F. Add 50 ml of developer solution, quickly swirl the

gel, and pour off developer. Repeat once more.

F.

Add 100 ml of developer and agitate until the
bands are visible. Be sure to stop the development
before the background becomes significant by
neutralizing the solution with 5 ml of stop solution.
Alternatively, pour off developer and add 100 ml
Destain solution II.

G. Wash the gel in 2–3 changes of deionized water.

Keep the gel in Destain solution II or dry it for
permanent storage.

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Содержание SE 400

Страница 1: ...user manual SE 400 SE 410 um SE400 IM Rev A0 06 04 Hoefer SE 400 SE 410 the Sturdier vertical slab gel electrophoresis units...

Страница 2: ......

Страница 3: ...tions precast and self cast gels 7 2 1 2 preliminary casting steps 8 2 2 Acrylamide gel preparation 2 2 1 resolving gel 11 2 2 2 stacking gel 12 2 2 3 gradient gel 13 2 3 Sample preparation 15 2 4 Fin...

Страница 4: ...for operating maintaining and servicing this product Utiliser seulement un g n rateur marqu CE ou dont la s ret a t certifi e par un laboratoire de test nationalement reconnu Eteindre le g n rateur a...

Страница 5: ...d separately is cast in the casting stand side of the unit The size of the gel is 14 15 cm if using the SE 400 and 14 23 cm if using the SE 410 After casting the sandwich is transferred into the lower...

Страница 6: ...turn the unit This unit is partially assembled to protect components during shipping To disassemble 1 Position the unit so that the electrical connectors face you 2 Note the holes at each side on the...

Страница 7: ...upper buffer chamber glass plates 2 casting cradle leveling feet 4 clamps lower buffer chamber Fig 1 SE 400 series main components Included but not shown Cams GelSeal grease 1 4 oz Spacer Mate Wonder...

Страница 8: ...SE 410 are 24 cm long A notched divider plate ordered separately can be used to run two gels at the same time Clamps Two 16 cm clamps are required to secure the 16 cm long sandwich These and an addit...

Страница 9: ...0 75 1 0 and 1 5 mm Combs May be ordered separately Teflon combs that form 1 to 28 wells are available in three thicknesses 0 75 1 0 and 1 5 mm Blank combs form a single large well and preparative com...

Страница 10: ...Altitude up to 2000 m Installation category II Pollution degree II Dimensions w h d SE 400 24 28 15 cm 9 5 11 6 in SE 410 24 36 15 cm 9 5 14 2 6 in Product certifications EN61010 1 UL3101 1 CSA C22 2...

Страница 11: ...ard precast gels purchased from commercial suppliers as well as self cast gels which can be prepared using the built in casting stand To cast multiple 14 16 cm gels the Multiple Gel Caster Kit which h...

Страница 12: ...t the top place a spacer along each edge and lay the second glass plate on top 4 Secure the sandwich with clamps Slide one clamp at a time along the sandwich sides Finger tighten one screw on each cla...

Страница 13: ...he laminated gasket into the casting cradle with the foam side down Place the glass plate assembly in the casting cradle screw side facing out see Fig 5 24 cm plates Place the sandwich so that the sho...

Страница 14: ...gth spacers leveling feet 4 casting cradle cams 2 insert the cam s in the cam holes and turn toward the center to lock the glass plate assembly gasket foam side down Fig 5 Caster components and assemb...

Страница 15: ...sandwich taking care not to trap air bubbles under the teeth 2 gel sandwich Pipet the solution into both sandwiches filling each to the same level below the notched edge Stacking gel Fill solution to...

Страница 16: ...ster Stacking gel resolution is optimal when prepared just before electrophoresis 1 Remove the overlay by rinsing the top of the gel several times with distilled water Invert the caster to drain To en...

Страница 17: ...less than the spacer thickness Place the cannula so that it rests at the bottom of the sandwich midway between the spacers 3 Prepare the monomer solution Calculate the total volume needed Prepare one...

Страница 18: ...e to oxygen Slowly deliver the overlay solution from a glass syringe fitted with a 22 gauge needle Apply the solution near the spacer and allow it to flow across the surface unaided 7 Allow the gel s...

Страница 19: ...verting the gel sandwich or caster Fill each well with electrophoresis buffer 2 Prepare the sample Increase liquid sample density with 10 glycerol or sucrose Add a tracking dye such as phenol red brom...

Страница 20: ...from the caster by removing both cams Clean away any gel adhering to the exterior of the gel sandwich Install the sandwich in the lower buffer chamber clamp screws facing toward the leads 3 Carefully...

Страница 21: ...ce the upper chamber onto the sandwich assembly then insert the cams into the cam holes ridge short end pointing down To secure the assembly turn the cams a full 180 so that the ridge points up not sh...

Страница 22: ...r into the upper chamber directing the buffer stream against the wall to avoid disturbing the samples Inspect the installation for leaks Fill both chambers the final volume for each chamber is 350 ml...

Страница 23: ...roperly check the position of the lower electrode shield which must clear the connectors and rest in the lower buffer chamber in front of the the shield guides Once all guides are in place press gentl...

Страница 24: ...ting and band distortion Table 3 lists starting point guidelines and adjustments for gel thickness number of gels and migration rate Table 3 Laemmli buffer system starting point guidelines Gel thickne...

Страница 25: ...or the SE 400 model and a Laemmli discontinuous buffer system The final voltage is typically 200 325 V depending on the length of the gel See Table 3 on page 20 Time A run is complete when the trackin...

Страница 26: ...ems such as current leaks incorrect buffer concentrations high salt concentrations or inconsistent chemical quality Check band progress after 5 minutes and again after an hour noting the migration rat...

Страница 27: ...e clamps from the sandwiches and remove Gently loosen and then slide away both spacers Use the Wonder Wedge plate separator tool to separate the plates 4 Carefully lift off one glass plate Handle the...

Страница 28: ...ses to clean any plastic part Do not soak the gaskets Clean with a mild detergent and allow to air dry Handle the safety lid with care to prevent damage to the electrode connectors Clean glass plates...

Страница 29: ...b must be removed add more monomer solution before reinserting the comb Incomplete or delayed Allow acrylamide gels to set for a minimum of 1 h polymerization Debris in wells Rinse out unpolymerized g...

Страница 30: ...ent leakage around gel Check for leaks all plates and spacers must be aligned and free of grease and cracks Sample or reagent preparation If the required pH of a solution is overshot do not back titra...

Страница 31: ...separation at a lower current or voltage setting to reduce Joule heating Reagent quality Use only the highest quality reagents Poor stacking Use only gels that were recently prepared Add a stacking g...

Страница 32: ...ng Pour a taller stacking gel For best results allow a stacking gel height of 2 5 times the height of the sample in the well Reagent quality Dispose of outdated acrylamide solutions and use only the h...

Страница 33: ...lycine Accordingly the resolving gel and the stacking gel contain Tris Cl buffers of different concentration and pH and the electropho resis buffer contains Tris glycine All buffers contain 0 1 SDS Po...

Страница 34: ...ris base FW 121 1 0 5 M 30 3 g HCl to pH 6 8 Deionized H2O to 500 m Store up to 3 months at 4 C in the dark 4 10 SDS solution 100 ml SDS FW 288 4 0 35 M 10 0 g Deionized H2O to 100 ml Store up to 6 mo...

Страница 35: ...n the gel 7 2X Sample treatment buffer 0 125 M TrisCl 4 SDS 20 glycerol 0 2 mM DTT pH 6 8 10 ml 0 5 M Tris Cl pH 6 8 Solution 3 0 125 M 2 5 ml 10 SDS 0 35 M Solution 4 0 14 M 4 0 ml Glycerol FW 92 09...

Страница 36: ...ue R 250 FW 826 0 3 mm 0 5 g Methanol Stir until dissolved 40 v v 800 0 ml Acetic acid 7 v v 140 0 ml Deionized H2O to 2 0 liters 10 Destain solution I 40 methanol 7 acetic acid 1 liter Methanol 40 v...

Страница 37: ...um carbonate Bring to 2 L with distilled water store in glass container 16 Developing solution 3 sodium carbonate 0 019 formaldehyde 200 ml of 3 sodium carbonate 100 l of 37 formaldehyde Prepare just...

Страница 38: ...a deep blue color until clear background results C Store the gel in Destain solution II For a more sensitive method silver stain protocol is recommended Silver Stain Protocol adapted from Morrissey 1...

Страница 39: ...on directly to the gel Shake gently for 30 minutes and then rinse the gel for 1 2 seconds with deionized water F Add 50 ml of developer solution quickly swirl the gel and pour off developer Repeat onc...

Страница 40: ...oln 3 1 25 ml 10 SDS Soln 4 0 3 ml 0 3 ml 0 3 ml 0 3 ml 0 05 ml Deionized H2O 14 6 ml 12 1 ml 9 6 ml 7 1 ml 3 00 ml 10 APS Soln 5 150 l 150 l 150 l 150 l 25 l TEMED 10 l 10 l 10 l 10 l 2 5 l Final Vol...

Страница 41: ...Struhl eds 10 6 1 10 6 8 1991 Denaturing gel systems Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T Nature 227 680 685 1970 Matsudaira P T and Burgess D...

Страница 42: ...rch Biochem Biophys 126 155 1968 Two dimensional electrophoresis Adams L D and Gallagher S R Two Dimensional Gel Electrophoresis Using the O Farrell System Current Protocols in Molecular Biology pp 10...

Страница 43: ...pper buffer chamber 1 SE4008B Blank silicone rubber gasket for casting stand 1 SE4009 Lid with electrodes for SE 400 16 cm 1 SE4156 Lid with electrodes for SE 410 24 cm 1 SE416 Lower buffer chamber ca...

Страница 44: ...1 1 SE511 20 1 5 28b 0 75 2 7 1 SE511 28 75 28b 1 00 2 7 1 SE511 28 1 0 28b 1 50 2 7 1 SE511 28 1 5 a Also for use with the Hoefer PR 200 Deca Probe Incubation Manifold b Comb depth 15 mm all others 2...

Страница 45: ...rs Order combs and spacers separately For up to 4 gels Gel Caster Kit 4 gels 18 16 cm 1 SE675 Includes 8 glass plates 3 space saver plates 5 filler sheets 100 sheets of wax paper Spacer Mate alignment...

Страница 46: ......

Страница 47: ...the USA Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA www hoeferinc com Coomassie is a trademark of ICI plc RBS 35 is a trademark of Pierce Chemical Co Teflon is a trademark of E I du Pont...

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