Items Tagged ‘Fero-Bacteria’

June 2nd, 2016

Plasmid Miniprep

By UNM CC UNM CC

M.Fero — 10/26/99 Materials Solution II: 0.2N NaOH/1% SDS Solution III: 3 M KOAc, pH4.8 RNAseA (DNAse free) 10 µg/mL Chloroform/Isoamyl alcohol (1/25 v/v) Isopropanol 70 % ethanol 13% PEG8000 Procedure Culture 5 mL of bacteria o.n. in LB + 100 mg/mL ampicillin. Spin down 1.5 mL of each culture (5,000 rpm x 3 min.) […]

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June 2nd, 2016

Ligation Optimization

By UNM CC UNM CC

The following protocol can be used to optimize ligation conditions for difficult to clone (e.g. very large) fragments. The principle is to independently characterize the ligation kinetics of the vector and insert DNA fragments and then to combine them in optimal ratios. The final ligation is also performed in two stages to optimize the proportion […]

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June 2nd, 2016

Large Scale Plasmid Preps: Qiagen/Cesium Method

By UNM CC UNM CC

M. Fero — 3/2003 Most plasmids can be adequately prepped by kits containing DNA binding columns. These columns do not do a great job of separating plasmid DNA from contaminating bacterial chromosomal DNA. If the chromosomal DNA is not sheared most of it will pellet along with the bacterial cell wall. However the relative purity […]

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June 2nd, 2016

Large Scale Plasmid Preps: PEG method

By UNM CC UNM CC

M. Fero — 12/29/00 Grow 250 ­ 500 mL of bacteria overnight in LB with 50 µg/mL of ampicillin. Transfer culture to Nalgene bottles. Spin in SGA rotor at 6000 rpm, at 4°C for 10 min. Discard supernatant. Keep everything on ice. Resuspend bacterial pellets by adding 15 mL of water and split into 2 […]

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June 2nd, 2016

Colony Hybridization

By UNM CC UNM CC

M.Fero — 11/94 Procedure Prepare serial 10-fold dilutions of transformed bacteria in LB and spread 100 µL onto LB/Amp plates, as described in the Electroporation protocol. Incubate at 37°C overnight. Also streak out positive and negative control bacteria on a separate plate. Select plates that have an optimal density of bacteria (i.e. 2 mm spacing […]

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June 2nd, 2016

Transformation of Electrocompetent E. coli with Blue/White Selection

By UNM CC UNM CC

M. Fero — 4/04 NOTE: add links once the posts are up. Procedure Desalt DNA template by EtOH precipitation in NaOAc followed by at least 2x washes with 70% EtOH. Resuspend in 5 – 15 µL of sterile H2O. Rinse cuvettes (if they have been used before) 5x with deionied H2O, and place them on […]

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June 2nd, 2016

Preparation of Electrocompetent Bacteria

By UNM CC UNM CC

8/17/2011 Shyamala Mohan Protocol adapted from Molecular cloning 3rd edition, Joseph Sambrook and David W. Russell Electrocompetent cells are prepared by growing cultures to mid log phase, washing the bacteria at low temperature and resuspending them in a solution of low ionic strength containing glycerol. The starting material can be a vial of compentent cells […]

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June 2nd, 2016

General Cloning Protocols

By UNM CC UNM CC

M.Fero 11/30/2011   NEED TO ADD links to other protocols once they are entered. Large Scale Preps (See Large scale plsasmid prep protocol for more details) Cultures: Inoculate a 5 mL LB/Amp (50 – 100 µg/mL) culture in early a.m. with a single colony. Use all 5 mL to inoculate a 500 mL LB/Amp culture […]

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Tags: Fero-Bacteria