• 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.
• 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.

Priprava poliakrilamidnih gelov za SDS-PAGE:

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

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, dH₂O 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.

To je verzija 1.01. Prosim za pripombe in dopolnitve. Hvala. Marko 21.11.95.
Popravljena napaka pri kolicini SDS v nanasalnem gelu: 8.9.98.

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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 supernatant; vortex vigorously
• add 1 ml absol. EtOH; invert the tubes 3x and centrifuge 10 min.
• discard supernatant and wash pellet with 200 ul 70% EtOH
• 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.

Flow chart (for expert users):

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 -> 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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