Polony PCR Amplification

This section outlines the protocol for single-molecule amplification within the acrylamide gel.  The protocol given here may differ in specifics from what is presented here, but reflects what we are doing currently (Summer 2003).  We have tried to include excessive detail here but let us know if anything is unclear.  The general steps are:

1.    Cast acrylamide gels
2.    Diffuse in PCR reagents
3.    PCR amplification
4.    Slide clean-up


Further Protocol 

Long PCR Protocol Reagents and Guidelines

General Guidelines for Long PCR Conditions and Enzyme Mixtures
Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Following the results of Cheng et al. (1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases have been used successfully for many applications. The buffer listed below works well with Tth and Vent, but not with others. If you are interested in using other polymerases make sure that you use compatible buffers. Error rates for other polymerases can be found at http://research.nwfsc.noaa.gov/protocols/taq-errors.html 

Read Full Protocol Here

Qiagen RNeasy Plant RNA Isolation

RNA isolation

RNeasy Kits are designed to isolate total RNA from small quantities of starting material. They provide a fast and simple method for the preparation of up to 100 µg total RNA from animal cells and tissues, bacteria, and yeast (RNeasy Mini Kits) or plant cells and tissues and filamentous fungi (RNeasy Plant Mini Kits). RNeasy Plant Mini Kits are fast and avoid tedious methods, such as CsCl step-gradient ultracentrifugation and alcohol precipitation steps, or methods involving the use of toxic substances such as phenol and/or chloroform. The purified RNA is ready for use in standard downstream applications such as RT-PCR, Northern, dot, and slot blotting, poly A+ RNA selection, primer extension, RNase and S1 nuclease protection, cDNA synthesis, differential display, expression-array and expression-chip analysis.

Further Protocol Information

Formaldehyde Agarose Gel Electrophoresis Protocol

The following protocol for formaldehyde-agarose gel electrophoresis gives enhanced sensitivity for gel and subsequent analysis (e.g. northern blotting). A key feature is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols (e.g. Sambrook, J. et al., eds. (1989) Molecular cloning — a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

1.2% Formaldehyde Agarose gel preparation

To prepare Formaldehyde Agarose gel (1.2% agarose) of size 10 x 14 x 0.7 cm, mix:

1.2 g agarose

10 ml 10x Formaldehyde Agarose gel buffer (see composition below)

Add RNase-free water to 100 ml

If smaller or larger gels are needed, adjust the quantities of components proportionately. Heat the mixture to melt agarose. Cool to 65°C in a water bath. Add 1.8 ml of 37% (12.3 M) formaldehyde (toxic) and 1 µl of a 10 mg/ml ethidium bromide (mutagenic) stock solution. Mix thoroughly and pour onto gel support. Prior to running the gel, equilibrate in 1x Formaldehyde Agarose gel running buffer for at least 30 min.

RNA sample preparation for Formaldehyde Agarose gel electrophoresis

Add 1 volume of 5x loading buffer per 4 volumes of RNA sample (for example 10 µl of loading buffer and 40 µl of RNA) and mix.

Incubate for 3–5 min at 65°C, chill on ice, and load onto the equilibrated Formaldehyde Agarose gel.

Gel running conditions

Run gel at 5–7 V/cm in 1x Formaldehyde Agarose gel running buffer.

Composition of Formaldehyde Agarose gel buffers

10x Formaldehyde Agarose Gel buffer

200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)

50 mM sodium acetate

10 mM EDTA

pH to 7.0 with NaOH

1x Formaldehyde Agarose Gel Running Buffer

100 ml 10x Formaldehyde Agarose gel buffer

20 ml 37% (12.3 M) formaldehyde

880 ml RNase-free water

5x RNA Loading Buffer

16 µl saturated aqueous bromophenol blue solution†

80 µl 500 mM EDTA, pH 8.0

720 µl 37% (12.3 M) formaldehyde

2 ml 100% glycerol

3084 µl formamide

4 ml 10 x Formaldehyde Agarose gel buffer

RNase-free water to 10 ml

Stability: Approximately 3 months at 4°C

Ref

2M H2SO4 for 100ml

2M H₂SO₄ for 100ml

• mix 50ml Milli Q water + 50ml 4M H₂SO₄ solution

4M H2SO4 for 1 liter

4M H₂SO₄ for 1 liter

• Carefully pour 392ml H₂SO₄ {Sulphuric acid 96% H₂SO₄ Carlo Erba reagents Code no. 410301 (Reanal 17769) FW 98.078} into 608ml Milli Q water (always pour acide into water)

TMB-buffer for 1 liter

TMB-buffer for 1 liter[0.1M Na-acetate, pH 5.5]

• Disolve in 950ml Milli Q water
• 13.6g Na-acetate {Nátrium acetát, kristályvizes CH₃COONa*3H₂O M:136.08 Reanal 14021}
• Adjust pH to 5.5 with app. 700ul cc. Acetic acid {Ecetsav 96% Reanal 0914-1-08-65 M:60.05}
• Adjust volume to 1 liter with Milli Q water