Lab Solutions and Reagents

most information adapted from:

Early Development of Xenopus Laevis: A Laboratory Manual.[1]

2000; (First ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Sive, Hazel (Author), Grainger, Robert M (Author), and Harland, Richard M (Author).

Additional methodology adapted from Khokha lab protocols [2]

click each bulleted list heading to view solution/reagent recipes

• culture media

Modified Barth's Saline (MBS)

Anitbiotics

Gentamycin

Oxytetracycline

Penicillin

Streptomycin Sulfate

High-salt MBS

Amphibian Ringers

Modified Frog Ringers (MR)

Marc's Modified Ringer's (MMR)

Normal Amphibian Medium (NAM)

Holtfreter's Solution

• immunohistochemistry

MEMFA

Dent's Fix (Methanol/DMSO)

Bleaching solution (Mayor et al. 1995)

Goat Serum (GS)

PBT

DAB

H2O2

Secondary Antibodies Coupled to Horseradish Peroxidase

Murray's Clearing Medium

• in situ hybridization

Nucleotide Stocks

5x SP6 Buffer

MEMFA

PTw

Triethanolamine 0.1 M

PTw Containing 4% Paraformaldehyde

Hybridization Buffer

SSC, 20X

Denhardt’s solution, 100X

RNase A

RNase T1

4% BMB Blocking Reagent in MAB

Alkaline Phosphatase Buffer

Levamisol 1 M

NBT/BCIP

PVA Alkaline Phosphatase Buffer

Bouin’s Fixative

Bleaching solution (Mayor et al. 1995)

• fixatives

MEMFA

Bouin’s Fixative

Dent's Fix (Methanol/DMSO)

Paraformaldehyde(PFA), 20%

PTw Containing 4% Paraformaldehyde

• DNA/RNA preparation
• embryo manipulation/dissection
• microinjection
• general buffers and salt solutions

HEPES 1 M, pH 7.4

1M MgCl2

10x Phosphate Buffered Saline (PBS)

20x Phosphate Buffered Saline (PBS)

PBT

Proteinase K(PK), 10 mg/ml

PTw

4M NaCl, 1 liter

SDS 10%

TAE 50X

1M Tris pH 9.5

• benzocaine stock, 100x (Khokha lab)

• high-salt solution for egg laying (Zorn lab/Ron Laskey)

• recipes for miscellaneous salts, solutions, etc. (Khokha lab)

agarose plates 1% - for use with laevis or tropicalis

Alkaline Phosphatase (AP) Buffer, with Levamisol and Tween

amphibian Ringers, 5 gal (19 L)

bleaching solution (Sive et al. 2000, Mayor et al. 1995)

4% BMB blocking reagent in MAB

Bouin’s fixative

cysteine, 2.0%

dehydrating embryos

Denhardt’s solution, 100X

dNTPs 10 mM

2.5% Ficoll

2.5% Ficoll in 1/3X MR (for use with X. laevis embryos)

3% Ficoll in 1/9XMR (for use with X.tropicalis embryos)

gentamicin stock, Sigma (1000X, 100 mg/ml)

HEPES 1 M, pH 7.4

hybridization buffer

levamisol 1 M

MAB, 10X

1M MgCl2

MEMFA* (Sive et al. 2000)

100X MR salts

1/3X Modified Ringers

1/9X MR + 1% Gentamicin

1/20X MR

modified Ringers

paraformaldehyde(PFA), 20%

phosphate buffered saline (PBS), 10X

proteinase K(PK), 10 mg/ml

PTw

rehydrating embryos (for embryos stored in 100% methanol)

RNase A (Sigma R 5000)

RNase T1 (Sigma R8251)

4M NaCl, 1 liter

SDS 10%

Sodium Phosphate Buffer(1M)

SSC, 20X

TAE 50X

Triethanolamine 0.1 M (pH7.0-8.0)

1M Tris pH 9.5