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Semidry Electroblotter

2-3

Thermo Scientific

Section 2

Setting Up

DNA Gels

Preparing a sequencing gel for transfer on the HEP-3

1. Remove the gel cassette from the gel box and place under cold,

running tap water until the surfaces of both glass plates are cool. This
facilitates handling of the gel and prevents the gel from "curling" as it
cools once it is removed from between the glass plates.

2. Place the glass plate sandwich, with the notched or shorter plate facing

up, flat on paper towels on the lab bench and remove any excess liquid
and remaining tape (or binder clips).

3. Remove one side spacer (wearing protective gloves) and insert a long

metal spatula between the glass plates where the spacer had been
present. Gently rock the spatula, forcing separation of the plate from
the gel. The gel will normally remain affixed to the bottom (not
notched) plate. Remove the top (notched) plate by slowly lifting it
from the side with the inserted spatula and gradually increasing the
angle lmtil the top plate is completely separated form the gel. If the gel
sticks to the top plate in an isolated spot, a stream of water from a
wash bottle can be sprayed at the spot to aid separation. Occasionally,
a gel sticks to the top plate instead of the bottom plate. In this case,
flip the gel sandwich over and follow the same procedure.

4. Once the plates are separated, remove the second side spacer along

with any extraneous bits of acrylamide around the gel. If excess water
was utilized to aid in the separation of the gel from the glass plate, use
a paper towel to absorb the excess liquid. Using a piece of dry blotting
paper(Whatman 3MM chromatography paper, 46 x 57cm), gently roll
it onto the gel beginning at one end and working slowly towards the
other end. Care should be taken to prevent air bubbles from forming
between the paper and the gel. Once the blotting paper is in position,
set another glass plate on top of the paper. Flip the entire sandwich
over. The gel should now be resting on top of the filter paper between
the two pieces of glass. Position the sandwich with one long side of the
glass hanging about one-third of the way off of the counter top. Slowly
push the bottom plate back toward the counter top, holding the top
plate in place, which will allow the blotting paper and gel to peel away
from the top piece of glass. The weight of the gel and blotting paper
causes the gel to slowly peel away from the top plate. Continue this
process until the gel is almost entirely peeled away from the glass. A
small portion of the gel may still be attached to the glass; in this case, a
small stream of water from a wash bottle can be used to aid in removal
of the gel.

Содержание OWL HEP-1

Страница 1: ...Semidry Electroblotter Models HEP 1 and HEP 3 Operating and Maintenance Manual 7007332 Rev 0 Visit us online to register your warranty www thermoscientific com warranty...

Страница 2: ...Thermo Scientific MANUAL NUMBER 7007332 0 5 1 12 Transfer to Marietta was The Panther 3 2003 ccs REV ECR ECN DATE DESCRIPTION By Preface Semidry Electroblotter i...

Страница 3: ...ngs found on the lower buffer chamber s This system is designed to meet IEC 1010 1 safety standards IEC 1010 1 is an internationally accepted electrical standard for laboratory instruments Statement o...

Страница 4: ...nt that has been put on the market after 13 August 2005 This product is required to comply with the European Union s Waste Electrical Electronic Equipment WEEE Directive 2002 96 EC It is marked with t...

Страница 5: ...can fill your needs for spare or replacement parts or provide you with on site service We can also provide you with a quotation on our Extended Warranty for your Thermo Scientific products Whatever Th...

Страница 6: ...wich Assembly 2 4 Using the System 3 1 Running the Blot 3 1 Transfer settings 3 1 Running Conditions 3 2 Factors That Affect Transfer Efficiency 3 2 Troubleshooting 4 1 Technical Tips 5 1 Semidry Blot...

Страница 7: ......

Страница 8: ...nsfer at low voltages without external cooling systems Plate electrodes also provide a uniform electric field for efficient even transfers Before starting unpack the unit and inventory your order If a...

Страница 9: ...Heavy Duty Knobs HEP 1 3 HEP 3 4 Power Supply Leads Lid Anode Cathode Base Figure 1 1 Exploded View Table 1 1 Parts List 1 base with stainless steel cathode 1 lid with platinum titanium anode 1 attac...

Страница 10: ...modifiers often use in protein transfer buffer These components however are antithetical in their effects both in terms of movement and adsorption Methanol restricts protein movement from the gel but...

Страница 11: ...l transfer protocol following separately DNA RNA If these gels were not run in IXTBE they should be equilibrated for 10 minutes in this buffer Protein Gels After electrophoresis remove the gel assembl...

Страница 12: ...f the bottom plate In this case flip the gel sandwich over and follow the same procedure 4 Once the plates are separated remove the second side spacer along with any extraneous bits of acrylamide arou...

Страница 13: ...clipping a comer of the membrane or using a ball point pen Clip the same comer until you retire 3 Wet the membrane according to its manufactures recommendations followed by a quick equilibration in t...

Страница 14: ...the filter stack 9 Add a few mL of buffer to the gel and gently layer the membrane as you did the gel 10 Repeat with three more pieces of filter paper 11 Holding the stack drain off all excess buffer...

Страница 15: ......

Страница 16: ...transfer down to say coincide with the setting up of a probe simply decrease the current mA to match the added time you require mA hr Std setting mA hr New setting Alternatively the current can be inc...

Страница 17: ...r efficient transfer of a specific molecule Factors That Affect Transfer Efficiency Protein HEP 3 Sequencing gel see HEP 1 set tings for other size gels on this unit DNA RNA Filter Paper FP 1 20 x 20c...

Страница 18: ...m and anode on top This means that an upward transfer is being performed rather than downward Follow the instructions care fully when assembling the transfer sandwich The pH of the transfer buffer is...

Страница 19: ...Alcohol was not used to prewet the membrane PVDF is hydrophobic and requires a short soak in methanol prior to transfer Air spaces are interfering with contact between the gel and the membrane Roll a...

Страница 20: ...delines are HEP l 2mA cm2 of gel for 1 hour These guidelines are just a starting point and exact conditions have to be determined Different kinds of blotting Western Blotting is a blotting method for...

Страница 21: ...Eckerskorn Christoph and Lottspeich Friedrich Structural characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysi...

Страница 22: ...with a useful reference list It also includes a very useful troubleshooting guide for nucleic acid and protein blots with pictures of the problems description of symptoms and proposed solutions 1X Tr...

Страница 23: ...Electroblotting This buffer is used with the HEP 1 Semidry Electroblotter Final 1 X composition Anode 1 Buffer 0 3M Tris Base 20 MeOH pH 10 4 Anode 2 Buffer 0 025M Tris Base 20 MeOH pH 10 4 Cathode B...

Страница 24: ...0 8M Tris 1 18M borate 24mM EDTA pH 8 3 CAPS Buffer pH 11 This buffer can be used to improve transfer of some proteins 10mm CAPS 3 cyclohexylamino 1 1 0 methanol propanesulfuric acid adjust to pH 11 w...

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Страница 26: ...use ethanol or other organic solvents to clean these products Do not autoclave bake or microwave your unit Temperatures over 50 C can damage acrylic s Note If an RNase free electrophoresis system is d...

Страница 27: ...S Phenol 5 solution U Hydroxide 10 S Glycerine Heptane commercial grade S Soap solution Ivory S Ammonium Hydroxide concentrate S Hexane S Sodium carbonate 2 S Aniline D Hydrochloric acid 10 S Sodium...

Страница 28: ...f 100 FP 3 Blotting Filter Paper 9cm x 9cm pkg of 100 FP 4 Blotting Filter Paper 10cm x 10cm pkg of 100 FP 6 Blotting Filter Paper 12cm x 16cm pkg of 100 FP 7 Power Supply Leads PSL 5 Buffer Kit recom...

Страница 29: ......

Страница 30: ...t beyond the original warranty period The Technical Services Department must give prior approval for return of any component or equipment At Thermo s option all non conforming parts must be returned t...

Страница 31: ...or to the component part beyond the original warranty period The Technical Services Department must give prior approval for return of any component or equipment At Thermo s option all non conforming p...

Страница 32: ...Thermo Fisher Scientific 401 Millcreek Road Marietta Ohio 45750 United States www thermofisher com...

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