Search Transcription Factor Binding site (TFBS) in bacteria

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http://server.molgenrug.nl/index.php/tfbs-search

http://genome2d.molgenrug.nl/index.php/genome2d

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Search for Binding Sites of Regulatory Elements

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http://www.genetools.us/genomics/tfbs%20search.html

http://www.genetools.us/genomics/Databases.htm

GelRed & GelGreen – DNA Stains

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It is 10,000 x.

Buy 500 ul for 105 $.

Dilute it by water to 10x, that is, add 500 ml water to 500 ul.

Instead of using it as Precast or Post-staining protocols, mix 1 ul of GelRed with 5 ul of DNA and run it.

https://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/

The Online Bioinformatics Resources Collection (OBRC)

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The Online Bioinformatics Resources Collection (OBRC) contains annotations and links for 2458 bioinformatics databases and software tools.

http://www.hsls.pitt.edu/obrc/index.php

T4 DNA ligase recipe

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 2X Rapid Ligation Buffer:

132 mM Tris-HCl, 20 mM MgCl2, 2 mM DTT, 2 mM ATP, 15% PEG 6000, pH 7.6

 

For ligation of long DNA fragments, prepare the buffer below:

10X T4 DNA Ligase Buffer:

500 mM Tris-HCl, 100 mM MgCl2, 50 mM DTT, 10 mM ATP, pH 7.6

From: T4 DNA ligase Rapid

Then, follow the instruction in “TaKaRa Clontech DNA Ligation Kit for Long Fragments”

TaKaRa Clontech DNA Ligation Kit for Long Fragments

 

 

Rubidium Chloride Competent Cells Protocol

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rubidium-chloride-competent-cells-protocol

Keep bacteria medium on ice for 15 min and the centrifuge for 10 min 3500 RPM.

Add 10 ml TFB1 .

Buffer TFB1:

MW Per 50 ml
RbCl 120.9 0.6046 gr
MnCl2-4H2O 197.9 0.49475 gr
Potassium acetate 98.14 0.14721 gr
CaCl2-2H2O 147 0.0735 OR 0.5 ml (1 M)
Glycerol 7.5 ml
Acetic acid glacial

Adjust pH to 5.8 with 1M acetic acid. Be very careful as you approach 5.8; if the pH drops lower than 5.8, a black precipitate may form. Filter sterilize (0.2µM) and store at room temperature.

Some say keep in 4 degree and protect from light.

Note:  take care when titrating this solution; if you overshoot and try to bring the pH back up with hydroxide, the manganese will fall out of solution.

pH is 6.5 and after adding 2 ul acetic acid ,  pH is 5.8

Buffer TFB2:

Per 50 ml
MOPS 209.3 0.10465
RbCl 120.9 0.06045
CaCl2-2H2O 147 0.55125 OR 3.75 ml (1 M)
Glycerol 7.5 ml
KOH 1 M

Adjust the pH to 6.5-6.8 with 1M KOH. Filter sterilize (0.2µM) and store at room temperature.

Some say keep in 4 degree and protect from light.

pH is 3.8 and after adding 375 ul fresh KOH 1M, pH is 6.8

and

http://www.med.unc.edu/pharm/sondeklab/files/resource-files

from

http://www.promega.ca/~/media/files/resources/product%20guides/subcloning%20notebook/transforming_bacteria_row.pdf?la=en