in molecular biology are there book that explain why we use material in every step in that protocol or are there simple book for explain general protocol that often used in molecular biology ? example: In step 4 (bold sentence) , it explain about why we use the protocol but other step no explain,
1. Inoculate a single colony containing the plasmid of interest in 5 mL of LB medium and 50 μg · mL−1 of appropriate antibiotics, using an inoculation loop or needle. Culture the bacteria at 37°C for 7 h to overnight with shaking at 250 rpm. For convenience, when preparing multiple cultures, select isolated colonies with autoclaved toothpicks (cut into ∼1 cm pieces) and inoculate cultures by dropping the toothpick directly into the culture tubes. Number each colony and tube correspondingly to assure tracking. If necessary, reincubate the plate at 37°C for 3–5 h and store the plate at 4°C.
Notes: (1) Use sterile techniques. Handle the toothpicks with flamed forceps. Alternatively, sterile pipette tips can be used for inoculation. Wipe the shaft of the pipette clean with ethanol before use.
(2) Prolonged culture will increase cell density, but at the same time, the number of aged or dead
cells increases and reduced plasmid yields can result. Rich media, such as Terrific Broth (TB), may
be used to shorten the culture time required to reach the desired cell density.
2. Add 1 mL of the overnight culture to a microcentrifuge tube and centrifuge at 12,000 × g for 30 s. Remove the liquid and invert the tube on a paper towel to dry the bacterial pellet for 4 min.
3. Resuspend the pellet by adding 0.1 mL of ice-cold plasmid lysis buffer and vortex for 2 min. Incubate the tube for 5 min at room temperature. This step lyses the bacteria by hyperlytic osmosis and releases the DNA and other contents.
4. Add 0.2 mL of freshly prepared alkaline solution and mix by inversion. Never vortex. Incubate the tube on ice for 5 min. The function of this step is to denature the plasmid and chromosomal DNAs and proteins.
5. Add 0.15 mL of ice-cold potassium acetate solution. Mix by inversion for 20 s and incubate on ice for 5 min. The purpose of this step is to selectively renature the plasmid DNA. Some chromosomal DNA may be partially renatured and bound by proteins, which will be extracted by phenol/chloroform in later step
Hi Riki,
Molecular Cloning: A Laboratory Manual (3 volume set) are pretty popular Molecular Biology protocol books. It comes in 3-volume set, authored by Sambrook et al. The labs I worked all kept one set handy. I like them. There are plenty of information in the books, including many recipes for preparing frequently-used buffers. To me, they are very useful. Now, they are in the Fourth Edition. I used the first and second editions. I haven't checked out the 4th edition yet. Attached pictures showed the 3rd edition and the 4th edition (From Amazon).
http://www.amazon.com/Molecular-Cloning-Laboratory-Edition-Three/dp/1936113422/ref=dp_ob_image_bk
I copied the description of the 4th edition from the site:
"Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Please take a look at our book preview site at molecularcloning.com. Building on thirty years of trust, reliability, and authority, the fourth edition of Molecular Cloning is the new gold standard--the one indispensable molecular biology laboratory manual and reference source."
Hi Riki,
Molecular Cloning: A Laboratory Manual (3 volume set) are pretty popular Molecular Biology protocol books. It comes in 3-volume set, authored by Sambrook et al. The labs I worked all kept one set handy. I like them. There are plenty of information in the books, including many recipes for preparing frequently-used buffers. To me, they are very useful. Now, they are in the Fourth Edition. I used the first and second editions. I haven't checked out the 4th edition yet. Attached pictures showed the 3rd edition and the 4th edition (From Amazon).
http://www.amazon.com/Molecular-Cloning-Laboratory-Edition-Three/dp/1936113422/ref=dp_ob_image_bk
I copied the description of the 4th edition from the site:
"Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Please take a look at our book preview site at molecularcloning.com. Building on thirty years of trust, reliability, and authority, the fourth edition of Molecular Cloning is the new gold standard--the one indispensable molecular biology laboratory manual and reference source."
I also suggest Molecular Cloning: A Laboratory Manual by Sambrook et al. It is really a good book on that give an insight into Why and How we use different reagents in molecular biology.
Hi Rikki,
I agree with Yuan-Yeu Yau and Arvind. Molecular Cloning: A Laboratory Manual by Sambrook et al. is a pretty standard book for practical molecular biology. You will find it really helpful.
Best,
Jogender
WORKING WITH DNA--excellent books with function of each solution, comments on why when and what, problems solving to check your learning, trouble shooting etc.
I have personally learnt a lot from this..Its excellent!!
Sorry I don't have a soft copy to share.
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