Section on Neural Gene Expression

SUBCLONING PROTOCOL

I. Preparation of Vector

A. Cut 1µg DNA using appropriate restriction buffer and enzymes in 20µl.
B.Dephosphorylate vector by adding 2µl 10X CIP buffer and 1µl CIP. Incubate for 1 hour at 50°C (no matter what kind of ends the restriction enzyme produces). Add 2.5µl stop dye.
C. Purify from low melt agarose gel (see III). Redissolve in 100µl TE.
II. Preparation of Insert
A. Cut 1µg DNA using appropriate restriction buffer and enzymes in 20µl.
B. Optional fill-in: Add 0.5µl Klenow and 2µl dNTPs (5mM), incubate 15min at 25°C. (Can usually be performed in the restriction buffer. Extract next if to be dephosphorylated). Can be inactivated at 75°C for 10min.
C.. Purify from low melt or NuSieve agarose gel (see III). Redissolve in 20µl TE.
D. PCR fragments that contain a restriction site can be cut as in II.A. If to be inserted blunt,then can be phosphorylated and filled in simultaneously (add ingredients to tube with DNA or add DNA as part of water component. Make sure that DNA was not co-precipitated with tRNA-use glycogen instead):
5µl dNTPs (10mM)
5µl rATP (10mM)
5µl 10X T4 Pol buffer (700mM Tris, pH7.5, 100mM MgCl₂, 50mM DTT)
27µl water
4µl T4 DNA polymerase
4µl T4 polynucleotide kinase
Incubate 30min at 37°C
III. Extraction from gel
A. Cut out desired piece and add TNE to 0.5ml total plus 25-50µg glycogen.
B. Add 0.5ml Tris-buffered phenol and heat at 65-70°C to melt gel. .
C. Spin 10 minutes. Re-extract with 37°C phenol and spin 5 minutes.
D. Extract Sn with phenol/chloroform.
E. Extract Sn with chloroform plus 15µl 4M NaCl.
F. Spin 5min, save supernatant and add 2 volumes EtOH.
G. Place on ice for 10 minutes, then spin 10 min. Dry pellet. Resuspend DNA inTE.
IV. Ligation (Overnight)
A. Mix 5µl insert (or TE for control), 1µl cut vector, 1µl 10X ligation buffer, 1µl 1M DTT, 1µl 10mM ATP, and 1µl T4 DNA ligase. (Note: BRL Ligation buffer may be used instead).
B. Incubate at 12°C overnight.
V. Ligation (Rapid)
A. Mix 5µl insert (or TE for control), 1µl cut vector, 2µl Solution 2 (5X concentration of DNA dilution buffer) from Rapid Ligation Kit from Boehringer Mannheim, and water to 10µl. Mix well.***Note: Make sure contents of Solutions 1 and 2 are mixed thoroughly before use.
B. Add 10µl Solution 1 (2X concentration of T4 DNA ligation buffer) and 1µl Solution 3 (5U/µl T4 DNA ligase). Mix well and let sit at room temp. for 5 minutes.
VI. Transfection of cells
A. Thaw DH5a ("subcloning") cells from BRL on ice for 30 min..
B. Add 3µl ligation mix to 50µl cells. Place on ice for 30 min.
C. Heat shock at 37°C for 20 sec. Place on ice for 2 min.
D. Add 950µl S.O.C. medium to cells/ligation mixture. Incubate 1 hour at 37°C in shaker (not above 225 cycles per minute) in 15ml double-snap top tube.
E. Plate out 200µl on LB/Amp/X-gal plates.
F. Incubate overnight at 37°C.
VII. Materials

A. 10X CIP buffer:
10mM ZnCl
10mM MgCl₂
100mM Tris-HCl, pH 8.3

B. 10X Ligation buffer:

0.5M Tris-HCl, pH 7.8
100mM MgCl₂
10mM ATP
500µg/ml BSA (Fraction V) - optional

C. S.O.C. Medium:

20 mM glucose + S.O.B. medium (0.5 ml 2M glucose + 50 ml S.O.B.)

S.O.B. Medium -

10g Bactotryptone 2.%
2.5g Yeast extract (0.5% final)
1.25ml 4M NaCl (10mM final)
0.42ml 3M KCl (2.5mM final)
* 5ml 1M MgCl₂ (10mM final)
* 5ml 1M MgSO₄ (10mM final)
(*Add after autoclaving)
500ml total

D. LB plates:

10g bactotryptone, 5g bacto-yeast extract, 10g NaCl. Shake until dissolved. Adjust pH to 7 with 5N NaOH in 1 L. total volume. Add 15g bacto-agar. Autoclave 20min. Store in refrigerator. To 500 ml LB agar add: 1ml of 50µg/ml ampicillin (in water) and 1ml of 2% X-gal (in dimethylformamide). Temperature of agar must not be higher that 56°C when the ampicillin and X-gal are added.

The Section on Neural Gene Expression is in the Laboratory of Cellular and Molecular Regulation, Division of Intramural Research Programs is within the National Institute of Mental Health (NIMH) is a part the National Institutes of Health (NIH), and is a component of the U.S. Department of Health and Human Services.