• Preparation of standard Laemmli vertical SDS-PAGE . Written in Slovenian language, this is a test file to encourage other contributions to a departmental collection of protocols. For Mini-Protean PAGE see a separate table with volumes needed.
• CTAB minipreparation of plasmid DNA (according to DelSal et al.)
• Isolation of DNA from agarose gels using NA-45 membranes. In English; an old protocol, transcribed for WWW.
• Western blotting with Biometra semidry transfer apparatus (in English).

Priprava poliakrilamidnih gelov za  SDS-PAGE:

Za velike sisteme dodajaj v vrstnem redu iz tabele:

raztopina	koncna koncentracija PAG v locevalnem delu				nanasalni del
(volumni so v ml; utezni %)	10%	12%	15%	17.5%	3%
akrilamid  (30% aa & 0.8-1.1% bis-aa)	8.00	9.60	12.00	13.6	1.25
dH2O	11.60	10.00	7.60	6.00	5.70
3 M Tris/HCl pH 8.8	3.00	3.00	3.00	3.00	XXX
0.5 M Tris/HCl pH 6.8	XXX	XXX	XXX	XXX	2.50
10% SDS	0.24	0.24	0.24	0.24	0.10
1.5% APS	1.20	1.20	1.20	1.20	0.50
TEMED	0.012	0.012	0.012	0.012	0.008

Za Mini-Protean PAGE pa potrebujes polovicne kolicine glede na zgornjo tabelo (za 2 gela); ce delas s predpripravljeno raztopino akrilamida (37.5:1; npr. Serva 10681) pa delaj po spodnji tabeli:
 
 

raztopina	locevalni gel	nanasalni gel
	15%	~3%
akrilamid (37.5:1)	4.5 ml	0.5 ml
dest. voda	5.3 ml	3.0 ml
3 M Tris pH 8.8	1.5 ml	xxx
0.5 M Tris pH 6.8	xxx	1.25 ml
10% SDS	0.12 ml	0.05 ml
1.5% APS	0.60 ml	0.25 ml
TEMED	0.006 ml	0.004 ml

kolicine so vecje od minimalno potrebnih za ~25% pri locevalnem in ~50% pri nanasalnem gelu
 

Akrilamid: 250 ml raztopine pripravis tako, da zatehtas 75 g akrilamida in 2 g bisakrilamida (za vecjo zamrezenost gelov lahko povecas kolicino bisakrilamida do 2.75 g - v tem primeru uporabljas za elektroforezo manjso koncno koncentracijo poliakrilamida), dopolnis z destilirano vodo do 250 ml, segrejes, da se hitreje raztopi in filtriras skozi papirni filter v temno steklenico. Hrani v hladilniku 1-2 meseca. Akrilamid je kancerogen; pripravljaj ga v digestoriju!

3 M TRIS: umeri pH-meter (s standardom pH 9) pred pripravljanjem raztopine. Za 500 ml rabis 181.65 g TRIS baze; nastavi pH na 8.8 in upostevaj, da rabis precej HCl, zato ne raztapljaj v prevelikem volumnu vode. Natancen pH pufrov je bistven za uspesno locevanje proteinov.

0.5 M TRIS: Za 500 ml zatehtaj 30.3 g TRIS baze; nastavi pH na 6.8. Bodi natancen pri titriranju pufra! pH 6.8 je na spodnji meji pufranja, zato obstaja nevarnost pretitriranja. Zadnje kapjice HCl dodajaj zelo pocasi.

10% SDS: K 10 g SDS dodaj dH₂O do 100 ml. Hrani ga na sobni temperaturi poljubno dolgo. SDS v prahu drazi sluznico, zato ga zatehtaj v digestoriju.

APS (amonijev persulfat; najdes ga v hladilniku v prehodu levo zgoraj): Zatehtaj svezega - pripravi 10 ml (150 mg APS, dH ₂O do 10 ml), ostanek shrani v hladilniku (obstojno do najvec 1 tedna - ce uprabljas starega, zvecaj volumen pri pripravi gela). APS lahko tudi zmrznes; tako je obstojen se dalj.

TEMED: Smrdi, zato steklenicko takoj po odpipetiranju zapri.

Priprava delovne raztopine:

Odpipetiraj potrebne volumne raztopin (glej tabelo). Premesaj pred in po dodatku TEMEDa, ki inducira polimerizacijo. Takoj nalij v vnaprej pripravljen stekleni sendvic in pazi, da v tekocino med nalivanjem ne pridejo mehurcki. Spodnji (locevalni) del gela, ki ga nalijes kot prvega, prekrij s plastjo vode ( pribl. 5 x 0.2 ml; pipetiraj previdno, ob robovih). Polimeriziran gel lahko shranis cez noc v hladilniku in nalijes nanasalni del gela naslednje jutro. Pri vstavljanju glavnicka pazi na mehurcke, ki se radi ujamejo pod zepki.
___________

Elektroforezni pufer (10x):
Za 1 l zmešaj 30.3 g Tris baze + 144 g glicina + 10 g SDS. Ne nastavljaj pH (nekateri protokoli imajo napacen podatek, da mora biti pH 8.3). Pred uporabo razredci 1:10.

Nanasalni pufer (reducirajoc) (5x):
Za 20 ml zmesaj 0.73 g Tris baze, 5 ml glicerola, 5 ml 2-merkaptoetanola in 2 g SDS. pH naj bi bil 8.8 (nastavi s HCl). Dodaj za konico spatule bromfenol modrega. (Kolicine lahko nekoliko povecas, a kaj vec kot 6x pufra ti verjetno ne bo uspelo pripraviti...)

Raztopina za barvanje:
Receptov je veliko, navajam enega od bolj preprostih (drugi vsebujejo tudi triklorocetno in sulfosalicilno kislino). Za 500 ml zmesaj 2.5 g Servablau (ustreza barvilu Coomassie Brilliant Blue), 50 ml ocetne kisline, 200 ml metanola in dopolni z vodo do koncnega volumna.

Raztopina za razbarvanje:
Za 1 l rabis 100 ml ocetne kisline, 400 ml metanola in 500 ml vode. Raztopino po uporabi lahko regeneriras preko oglja (pazi, da oglje ne prehaja v filtrirano raztopino!); ko je ze stara, ji dolij malo metanola in ocetne kisline - gel ni tako obcutljiv, da ne bi prenesel 20% in vec odstopanja od recepture.
 

To je verzija 1.01. Prosim za pripombe in dopolnitve. Hvala. Marko 21.11.95.
Zadnja sprememba 25.11.99 .

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• collect 2 x 1.5 ml o/n culture by centrifugation (all centrifugation steps in a microcentrifuge at 14.000 rpm) for 1 min.
• resuspend pellet in 300 ul of STET (8% sucrose, 0.1% Triton X-100, 50 mM EDTA, 50 mM Tris pH 8.0)
• add 12 ul lysozyme (20 mg/ml in water; store the stock frozen) and incubate at room temp. for 5 min.
• incubate in boiling water bath for 75 sec, then centrifuge 10 min.
• discard the pellet (use a toothpick or pipet) and add 12 ul of 5% CTAB to the supernatant; vortex
• centrifuge 5 min.; discard supernatant
• add 450 ul of 1.2 M NaCl to the pellet; vortex vigorously
• add 1 ml absol. EtOH; invert the tubes 3x and centrifuge 10 min.
• discard supernatant; add 200 ul 70% EtOH and vortex
• centrifuge 2-5 min; discard supernatant
• dry the pellet (speedvac 2 min.); add 32 ul dH2O

Troubleshooting:

• too much chromosomal DNA in preparation: decrease boiling time
• low yield of plasmid or plasmid larger than 20 kb: possible contamination of your cultures
• DNA won't dissolve: speedvac for a shorter period of time; residual insoluble material contains little plasmid DNA and more of contaminants (RNA, chromosomal DNA, proteins)
• On agarose electrophoresis, RNA can smear down to the size of  ~1 kb DNA unless RNAse is added.

spin 2 x 1.5 ml culture -> pellet + 300 ul STET (vortex) + 12 ul lysozyme (vortex) -> 5 min RT -> boil 75 sec -> centrifuge 10 min -> discard pellets / add 12 ul CTAB (vortex) -> centrifuge 5 min -> discard supernatant / pellet + 450 ul NaCl (vortex) + 1 ml EtOH -> invert 3x, centrifuge 10 min. -> discard supernatant -> wash pellet with 200 ul 70% EtOH (vortex & centrifuge 2 min) -> discard supernatant -> speedvac 2 min -> dissolve in 32 ul dH2O

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NA-45 is a membrane form of DEAE cellulose. For ds DNA < 7 kb the recovery is typically 50-90%.

Literature:

Lizardi, P.M. (1981) Binding and recovery of DNA and RNA using S&S NA-45 DEAE membrane; Schleicher & Schuell Sequences, Application update Nr. 364

Materials:

NA-45 DEAE membrane (Schleicher & Schuell, order nr. 40-23410)
High salt NET buffer: 1 M NaCl, 0.1 mM EDTA, 20 mM Tris/HCl pH 8.0
water-staurated n-butanol, absol. ethanol, 70% ethanol
TE buffer

Procedure:

1. Perform agarose gel electrophoresis of DNA. Calculate the amount of DNA to be isolated - consider the binding capacity of the membrane which is approx. 7 ug/cm².
2. Cut the gel with a scalpel on each side of the band from which you intend to purify DNA; incisions can be broader than the actual band, so that no DNA will ecsape on the sides.
3. Dry the surface of the gel with a paper towel. Cut the NA-45 membrane to get the pieces which would fit into the incisions you made into the gel. Insert the membranes and take care that they reach the bottom of the gel. On the top, the membranes must not touch each other.
4. Continue electrophoresis until DNA binds onto the membrane (check under UV). Immediately (the membranes are not allowed to get dry; DNA might bind to it ireversibly) transfer the membranes with the desired fragments into microcentrifuge tubes and add 250 ul of the high-salt NET buffer.
5. Centrifuge to submerge the membranes into the buffer. Incubate at 60°C (55-68°C) for 30 min (10-45 min) with occasional swirling the tubes. Transfer the buffer into fresh tubes. Wash the membranes with another 150 ul of the elution (high-salt NET) buffer. Incubate at the above conditions for 5 min. Combine supernatants.
6. Remove ethidium bromide by extraction with 400 ul butanol: shake well and centrifuge for 2 min. Discard the organic phase.
7. Add absol. ethanol to top, invert the tubes several times and incubate at -20°C for 30 min (5 h).
8. Centrifuge 15 min. Wash pellet with 200 ul 70% ethanol. Centrifuge 3 min. Dry the pellet and redissolve in 10 ul TE buffer.

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Western blotting with Biometra apparatus

After the electrophoresis (PAGE), determine the dimensions of the gel with the samples for transfer. Standard full-size Mini-Protean gel is approx. 8 x 5 cm.
Cut the membrane (Nitrocellulose or PVDF) to the size of the gel (or the fragment containing your samples) and prepare 6 pieces of blotting paper of the same dimensions.
When using the PVDF membrane, wet it in 100% methanol for ~1 min, followed by 2 min incubation in the transfer buffer (see below). Blotting paper should be made wet in transfer buffer immediately prior to the assembly of the blotting sandwich. The gel should be incubated in the same buffer for a few minutes, too.

Sandwich assembly:

Biometra apparatus has a graphite anode (+) on the bottom of the apparatus and cathode (-) on the top. Wipe the electrodes with distilled water/cleenex. Put on the bottom electrode 3 layers of wet blotting paper (one by one), eliminating any bubbles by rolling a pipette or a test tube over the layer. Onto the 3 blotting papers, lay the wet membrane, followed by the gel. Again, be sure there are no bubbles between the layers. Cover with another 3 layers of wet blotting paper, eliminate bubbles and cover with the cathode.
Apply ~40 mA (voltage would be ~ 4 V only; use the Biometra power supply)  for a full-size Mini-Protean (less for smaller sizes). Standard 10-15% PAG shall be blotted for about 1 h.
Dismount the sandwich, discard the blotting papers, stain the gel with CBB as usually and proceed with protein detection on the membrane either by immunostaining or CBB staining (exclude acetic acid from both staining and destaining solution; ATTENTION:  CBB staining is completed in ~1 minute !). For CBB staining for futher N-terminal amino acid sequence determination, the membrane can be washed o/n in distilled water at 4 deg. C prior to staining.

Blotting buffer:
Mix 2.93 g TRIS base, 5.81 g glycine, 3.75 ml 10% SDS and 200 ml MeOH. Fill to 1 l with distilled water and dissolve. No pH adjustment is required.
 

This is version 1.02 (March 2005).