Northern electroblotting

tfitzwater at gilead.com tfitzwater at gilead.com
Wed Nov 22 11:44:25 EST 2000


>Roger Rabbit (acdgteg at hgmp.mrc.ac.uk)
>Tue 21 Nov 2000 - 12:07:33 GMT


>Hi all,


>Does anyone have experience with electroblotting Northerns?


>Markus


Northern Protocol

Tim Fitzwater
Gilead Sciences

100x TAE buffer 5 blotting buffer for Northerns
|-----+-----------+---------+-----------+------------------------------|
|     | [1x]      | [100x]  |           |                              |
|-----+-----------+---------+-----------+------------------------------|
|     | 10 mM     | 1 M     | 121.14 g  | Tris® base                   |
|-----+-----------+---------+-----------+------------------------------|
|     | 0.5 mM    | 50 mM   | 18.61 g   | Na2EDTA                      |
|-----+-----------+---------+-----------+------------------------------|
|     | 5 mM      | 500 mM  | 41.04 g   | NaOAc-anhydrous              |
|-----+-----------+---------+-----------+------------------------------|
|     |           |         | ~43 ml    | glacial acetic acid to pH 7.8|
|-----+-----------+---------+-----------+------------------------------|
|     |           |         |x ml       |Type I water                  |
|-----+-----------+---------+-----------+------------------------------|
|     |           |         | 1000 ml   |                              |
|-----+-----------+---------+-----------+------------------------------|



Autoclave  or  filter  sterilize.  This buffer is used with the Hoeffer/LKB
apparatus  to transfer RNA to nitrocellulose or nylon.  The Hoeffer/LKB box
requires almost 5 liters of 1x TAE5, while the Bio-Rad unit requires 3.3 L.
Gels  do  not  need to be presoaked to remove urea.  Acetate is oxidized to
carbonate during electrophoresis, raising pH.

50x Denhardt's
|-------------+---------------+----------------------------------|
| 1%          | 5 g           | Ficoll                           |
|-------------+---------------+----------------------------------|
| 1%          | 5 g           | polyvinylpyrolidone              |
|-------------+---------------+----------------------------------|
| 1%          | 5 g           | acetylated BSA                   |
|-------------+---------------+----------------------------------|
|             |x ml           |Type I water                      |
|-------------+---------------+----------------------------------|
|             | 500 ml        |                                  |
|-------------+---------------+----------------------------------|



Dissolve  with  mild  heat  and  stir.   Spin  2000 rpm for 15 min.  Filter
sterilize  supernatant through 0.45 µm unit with 2 prefilters. 50x stock is
difficult  to  filter sterilize unless prefilters are used.  Store aliquots
at -20°C. Do not thaw at elevated temperatures.

20x SSPE
|---------------+--------------------------------|
| 175.3 g       | NaCl                           |
|---------------+--------------------------------|
| 27.6 g        | NaH2PO4                        |
|---------------+--------------------------------|
| 7.4 g         | Na2EDTA                        |
|---------------+--------------------------------|
| 800 ml        | Type I water                   |
|---------------+--------------------------------|
| 20.5 ml       | 10 N NaOH to pH 7.4            |
|---------------+--------------------------------|
| x ml          |Type I water                    |
|---------------+--------------------------------|
| 1000 ml       |                                |
|---------------+--------------------------------|



Autoclave or filter sterilize.  SSPE can replace SSC plus NaPO4, pH 6.7.

50% Dextran sulfate MW 5000
|---------------+--------------------------------|
| 2 g           | Dextran sulfate MW 5000        |
|---------------+--------------------------------|
| x ml          |Type I water                    |
|---------------+--------------------------------|
| 4 ml          |                                |
|---------------+--------------------------------|



Dextran  sulfate  MW  5000 works just as well as the traditional MW 500,000
material,  but  is significantly easier to dissolve.  Weigh dextran sulfate
directly  into  anti-static  treated  tube  (do  not  try  to transfer from
weighing paper or weigh boat).
Dextran sulfate supports the growth of bacteria.  Store at -20°C.



Northern blots


1.     A Hoeffer/LKB Transphor unit is recommended for Northern blots.  The
Bio-Rad unit has been known to melt when incorrect voltage was applied.
The  recommended  transfer  membrane  for  very  small RNA was 0.1 µm nylon
(Schleicher  &  Schuell  Nytran  or  ICN  Biotrans) but this material is no
longer  available.  RNA that is < 110 nucleotides will tend to go through a
0.2 µm membrane.  I am trying Schleicher & Schuell nitrocellulose membranes
(BA79/0.1 µm and BA75/0.05 µm).
Hoeffer  sponges  are  6"  x  9"  and  the  gel  must  be  cut to fit these
dimensions.
The  Hoeffer gel box can blot 4 gels simultaneously if additional cassettes
are purchased.
The Hoeffer unit generates more heat than the BioRad unit.
BioRad  sponges  are  6  1/8" x 8 3/8" and the gel must be cut to fit these
dimensions.
The BioRad box is limited to a single gel.
Polyacrylamide/TBE  gels  ranging from 4 to 8% have been successfully used,
with  or  without  urea.   It is not necessary to presoak the gel to remove
urea  prior  to blotting.  If the gel is severely overloaded, some RNA will
transfer to a second layer of nylon.

2.     Water and buffers do not need to be DEPC-treated if they do not come
from  carboys.  Carboy spouts tend to be a source of mildew RNases that are
resistant to DEPC.

3.     Prep a clean tray large enough to hold the blot cassette and soak in
0.1%  DEPC  or  3% hydrogen peroxide for 10 min at room temperature.  Cover
the pan during the soak.

4.     Prep 5000 ml 1x TAE5 for the Hoeffer unit or 3300 ml 1x TAE5 for the
BioRad unit.
Pre-chill the buffer on ice in the coldroom.

5.     Cut  nylon or nitrocellulose and 2 pieces of Whatman 3MM to required
dimensions  while  wearing  gloves  to prevent finger oils from coming into
contact with membrane.  Clip one corner to indicate orientation.

6.     Drain 0.1% DEPC or 3% hydrogen peroxide from the tray.  Rinse with a
trace  of  1x TAE5.  Add remaining portion of 1 liter 1x TAE5 to tray.  Add
plastic  gel  cassette  and  sponges.   Squeeze the air out of the sponges.
Soak the membrane briefly by laying it on the sponges.

7.     Open the gel plates.  Apply membrane to gel and gently brush out air
bubbles.   Dip  one  piece of 3MM very briefly in 1x TAE5 and lay on top of
membrane.   Remove  air bubbles.  Lay glass plate on 3MM and flip gel over.
Remove  the  other  plate  (this  seems  to  be  difficult)  and prewet the
remaining  piece  of 3MM.  Lay this on top of the gel.  Remove air bubbles.
Move to plastic cassette and assemble holder.

8.     Place  the  membrane  side  of  the  gel  cassette  towards  the red
electrode.  Add prechilled 1x TAE5 buffer to cover.  Use the 1x TAE5 buffer
in the presoak tray as part of the required buffer.

9.     Careful!   Electrode plugs on the BioRad Transblot will corrode when
wet.  Keep the buffer below the electrode plugs.

10.    Place a magnetic stir bar in the bottom of the box.  Move box to 4°C
coldroom.   Buffer  must  be  stirred  during  blotting  run.  Set magnetic
stirrer at speed 6.

11.   Hoeffer/LKB Transphor 10 v/30 min then 40 v/2 hours or 20 v/overnight
      BioRad  Transblot  20  v  overnight.  Do not use the 40 volt protocol
with the BioRad unit.
|-----------------+-----------------+-----------------+-----------------|
| Time            | Volts           | Amps            | Temperature     |
|-----------------+-----------------+-----------------+-----------------|
| Start time      |                 |                 |                 |
|-----------------+-----------------+-----------------+-----------------|
| Reduce voltage  |                 |                 |                 |
|-----------------+-----------------+-----------------+-----------------|
| Stop            |                 |                 |                 |
|-----------------+-----------------+-----------------+-----------------|



12.  UV fix the wet membrane (leave it on the wet 3MM support) with 120,000
µjoules UV via the UV crosslinker.  Alternatively, use a Sylvania
germicidal UV lamp exactly 12 cm from the bulb for exactly 2 min.  Caution:
nitrocellulose is highly flammable.  Air dry the membrane.  Place a single
membrane in a hybridization bag.  Seal the bag.  Cut one corner and add
prehyb solution at 5 mL/100 cm2 membrane.  Incubate in shaking waterbath
for 3 hours at 42°C.  Cut a corner of the bag and remove all prehyb.  Add
hybridization solution at 5 mL/100 cm2 membrane.
13.  Add 1 e 7 cpm 32P-labeled probe to 300 µl reserved hyb mix and boil
for 3-5 min.  Transfer the probe to ice and then add the probe to the
hybridization bag within 20 min.  Seal the bag.  Rinse the bag in the sink
to remove prehyb or hyb mix from the outside of the bag, as these will
support the growth of bacteria in the waterbath.  Incubate the hyb solution
in a shaking waterbath overnight at 55°C.  Alternatively, a rotisserie
apparatus can be used instead of hybridization bags and shaking water
baths.

13.   Prehybridization mix (add in order listed at 5 ml/100 cm2 membrane)
|----------+------+-------+-------+------+---------------------------|
| Conc.    | 5 mL | 10 mL | 15 mL | x mL | Stocks                    |
|----------+------+-------+-------+------+---------------------------|
| 5x       | 500  | 1000  | 1500  |      | 50x Denhardt's            |
|          | µl   | µl    | µl    |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 5x       | 1 ml | 2 ml  | 3 ml  |      | 20x SSC                   |
|----------+------+-------+-------+------+---------------------------|
| 50 mM    | 250  | 500 µl| 750 µl|      | 1 M NaPO4, pH 6.7         |
|          | µl   |       |       |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 50%      | 2.5  | 5 ml  | 7.5 ml|      | deionized formamide       |
|          | ml   |       |       |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 250 µg/ml| 250  | 500 µl| 750 µl|      | 5 mg/ml sheared salmon    |
|          | µl   |       |       |      | sperm DNA                 |
|----------+------+-------+-------+------+---------------------------|
| 1%       | 100  | 200 µl| 300 µl|      | 50% dextran sulfate MW    |
|          | µl   |       |       |      | 5000                      |
|----------+------+-------+-------+------+---------------------------|
|          | 350  | 700 µl| 1050  |      | Type I water              |
|          | µl   |       | µl    |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 0.1%     |50 µl | 100 µl| 150 µl|      |10% SDS                    |
|----------+------+-------+-------+------+---------------------------|
|          | 5 ml | 10 ml | 15 ml | x ml |                           |
|----------+------+-------+-------+------+---------------------------|

Prep  the  hyb  mix  and  prehyb  mix  fresh..  The hyb mix can sit at room
temperature during the 3 hour prehyb step.

14.   Hybridization mix (add in order listed at 5 ml/100 cm2 membrane)
|----------+------+-------+-------+------+---------------------------|
| Conc.    | 5 ml | 10 ml | 15 ml | x ml | Stocks                    |
|----------+------+-------+-------+------+---------------------------|
| 1x       | 100  | 200 µl| 300 µl|      | 50x Denhardt's            |
|          | µl   |       |       |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 5x       | 1 ml | 2 ml  | 3 ml  |      | 20x SSC                   |
|----------+------+-------+-------+------+---------------------------|
| 20 mM    | 100  | 200 µl| 300 µl|      | 1 M NaPO4, pH 6.7         |
|          | µl   |       |       |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 50%      | 2.5  | 5 ml  | 7.5 ml|      | deionized formamide       |
|          | ml   |       |       |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 100 µg/ml| 100  | 200 µl| 300 µl|      | 5 mg/ml sheared salmon    |
|          | µl   |       |       |      | sperm DNA                 |
|----------+------+-------+-------+------+---------------------------|
| 5%       | 500  | 1000  | 1500  |      | 50% dextran sulfate MW    |
|          | µl   | µl    | µl    |      | 5000                      |
|----------+------+-------+-------+------+---------------------------|
|          | 650  | 1300  | 1950  |      | Type I water              |
|          | µl   | µl    | µl    |      |                           |
|----------+------+-------+-------+------+---------------------------|
| 0.1%     |50 µl | 100 µl| 150 µl|      |10% SDS                    |
|----------+------+-------+-------+------+---------------------------|
|          | 5 ml | 10 ml | 15 ml | x ml |                           |
|----------+------+-------+-------+------+---------------------------|


15.    Wash  square Pyrex dish that will fit in shaking waterbath.  Soak in
3% hydrogen peroxide or 0.1% DEPC for 10 min.

16.   High Stringency Washes 1, 2 and 3
|---------+---------+---------+----------------------------------|
| Wash 1  | Wash 2  | Wash 3  |                                  |
| 2x SSC  | 1x SSC  | 0.5x SSC|                                  |
|---------+---------+---------+----------------------------------|
| 800 ml  | 40 ml   | 20 ml   | 25x SSC, pH 7.0                  |
|---------+---------+---------+----------------------------------|
| 910 ml  | 940 ml  | 970 ml  | Type I water                     |
|---------+---------+---------+----------------------------------|
| 10 ml   | 10 ml   | 10 ml   |10% SDS                           |
|---------+---------+---------+----------------------------------|
| 1000 ml | 1000 ml | 1000 ml |                                  |
|---------+---------+---------+----------------------------------|


17.    Discard  the  probe  and hybridization solution in 32P liquid waste.
Rinse  the  bag with approximately 30 ml wash 1 and discard the wash in 32P
liquid  waste.  Discard the 0.1% DEPC in the tray and add approximately 100
ml  wash  1  to the dish.  Transfer the membrane to the dish and cover with
Saran.   Shake  gently at room temperature for 10 min.  Discard wash in 32P
waste.   Microwave the remaining wash for 1 min to prewarm slightly and add
to  the Pyrex dish containing the probed membrane.  Incubate at 65°C for 30
min.   Washes  may be performed in the hybridization bag, but may result in
higher  background.  Discard  wash  1  and add 1000 ml prewarmed wash 2 and
incubate  at 65°C for 30 min. Discard wash 2 and add 1000 ml prewarmed wash
3 and incubate at 65°C for 30 min.

18.    Air  dry  the  filter  on 3MM paper and wrap with Saran.  Expose the
membrane as desired.

Reprobing

Do  not  let the membrane dry out if reprobing is required.  Place the blot
in  a  clear  plastic  sheet  protector  and  seal  it closed with tape for
chemiluminescent  exposure.   For  storage,  seal the blot in a water-tight
seal-a-meal  pouch  and  store  at  ?20°C.   Note  that  only  SaranWrap is
water-tight.   Other  plastic  wraps are water-permeable and allow blots to
dry  out  (and  allow  water from wet gels to contact autoradiography film,
causing black spots).

Charge

Neutral  membranes  have  an  equal  and  uniform  distribution of charges.
Positively  charged  membranes  have  a  preponderance  of positive charges
across  the  matrix.   They  will  hold  the  same  amount of nucleic acid.
Positively  charged  membranes  have stronger retention: the target remains
bound  longer.  UV crosslinking forms a covalent bond between nucleic acids
and  the  positive  charges  of the membrane.  Fixation by baking creates a
hydrophobic  interaction  between  the  nucleic acids and the membrane.  If
stripping  and  reprobing  is  required,  it is better to UV crosslink to a
positively  charged  membrane  in  order  to  preserve the integrity of the
target during the stripping step.

Sidedness

Quality  membranes  do  not  have  a correct side.  The charges are equally
distributed on both sides.

Porosity

Nominally,  0.22  µm  pore membranes have greater surface area than 0.45 µm
membranes.  They also require better blocking.  For Northern blots of small
RNAs (~ 100 nucleotides), 0.1 µm membranes are recommended.

Background

Particulates in unfiltered buffer, skin oils, nonspecific antibody binding,
addition of too much probe, inadequate post-hybridization washing, improper
wash temperature, and other factors can create background fog or spots on
membranes.


---






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