Real-time PCR Video for GM soybean DNA

PCR Animation

PCR Tutorial Animation

Moleclular Biology of the Gene Wattson

CD Moleclular Biology of the Gene, Wattson

This CD-ROM contains 20 interactive animations, 13 structural tutorials, and 19 critical thinking exercises. The media has been designed not only to reinforce fundamental concepts, but also to extend the content and scope of the text. Link, Password: chromatine

Large scale gene transfer between organisms

Large scale gene transfer between single-celled and multicellular organisms reported

Wolbachia has on some occasions inserted almost its entire genome into species that it infects reported scientists at the J. Craig Venter Institute and the University of Rochester. This is the first example of large-scale horizontal gene transfer between single-celled and multicellular organisms. Although horizontal gene transmission is common among single-celled organisms, it is rare among multicellular organisms, and large scale transfer like that of an entire genome had previously not been suspected.

The scientists found that in addition to Wolbachia engaging in almost complete genome transfer into Drosophila ananassae, it also had made significant transfer in 3 other insects species and 4 species of nematodes. The researchers found candidate species by scanning genetic databases for sequences found in Wolbachia. The scientists also found that these added sections were conserved by reproduction; that is the added sections stayed in the genomes after multiple generations. Moreover, there is evidence that suggests that the segments of Wolbachia's genome increased the reproductive fitness of the insect species.

The transfers likely occurred during attempts at DNA repair in which the repair mechanisms incorporated Wolbachia DNA (available since the cells were infected with Wolbachia) into the genomes. These results could have major implications for understanding of evolution. The research also has implications for various forms of sequencing research, since when sequencing species, bacteria sequences are frequently ignored as they are generally assumed to be contaminants rather than good data.

Wolbachia, a genus of bacteria that normally infects anthropods, especially insects, is already known for its odd behavior that can affect species in strange ways. For example, Wolbachia has been shown to be correlated with fast evolution among species it infects and is suspected for being responsible for a variety of speciation events as a side affect of Wolbachia creating reproductive barriers. Wolbachia by some estimates infects more than half of all anthropods and is already thought to play a major role in the evolution and speciation of many invertebrates.

Since Wolbachia can generally only reproduce through females, it has adopted a number of strategies that treat males and females of species differently that can result in reproductive barriers. These strategies include killing of males, forced parthenogenesis, and preventing infected males from reproducing with uninfected females.

WikiNews September 4, 2007

Molecular Biology Ebook Pseudomonas: Volume 4:

Pseudomonas: Volume 4: Molecular Biology of Emerging Issues

The first three volumes of the Pseudomonas series covered the biology of pseudomonads in a wide context, including the niches they inhabit, the taxonomic relations among members of this group, the molecular biology of gene expression in different niches and under different environmental conditions, the analysis of virulence traits in plants, animals and human pathogens as well as the determinants that make some strains useful for biotechnological applications and promotion of plant growth. pass: gigapedia Link

Protocols Methods in Molecular Biology Protein Sequencing

Protein Sequencing Protocols (Methods in Molecular Biology-Ebook)

ISBN: 0896033538
Title: Protein Sequencing Protocols (Methods in Molecular Biology, 1st edition, 1997-02
Author: Bryan John Smith (Editor)
Publisher: Humana Press
Link

LARGE SCALE PLASMID PREP CsCl METHOD

Triton lysis / CsCl method

Grow 500ml cultures with antibiotic selection shaking at 37oC.
When OD600 = 0.8 you may add chloroamphenicol to 125ug/ml.
Let it go overnight.

Spin down the cultures in sterile bottles at 5000rpm for 10 min at 4oC.
Make sure the seals on the bottle are properly seated and
the bottles are balanced within 0.1g.
Carefully decant the supernatant down the drain.
*You may replace the cap and store the pellet at -20oC.
*Before going on with the prep, be sure the ultracentrifuge and rotor are available
and sign up to use them.
You'll do one 30 min spin and one for 36 hours!

Prepare lysozyme on put on ice.
You'll need 0.5ml per culture.
Use plastic tube, and don't vortex.
For 1ml: 0.75ml water
0.25ml 1M Tris-Cl, pH 7-8
10mg lysozyme (stored in desiccator at -20oC)

If the pellet isn't soft , vortex it for a long time until it is.

Add to pellet 3ml 25% sucrose, 50mM Tris-Cl. Mix
Add 0.5ml lysozyme. Mix gently. Leave on ice for 5 min.

Add 1ml 0.5M EDTA, leave on ice for 5 min.

Add 4.5ml Triton juice. Mix gently.
[Triton juice: 4ml 10% Triton-X100, 20ml 0.5M EDTA, 20ml 1M Tris-Cl, 320ml water]
Pour the mess carefully into clean centrifuge tubes.
Put on ice and watch for at least 10 min for the solution to become viscous.
Take the cap off and see the snotty DNA.
Balance the tubes to within 0.01g.

Spin the tubes at 30,000rpm for 30 min at 4oC.
Make sure the rotor is clean and dry, and that the O-ring is seated properly.

Decant the supernatant into a 15ml disposable tube. Don't let pellet fall.
Estimate the volume by the gradations on the tube to the nearest 0.1ml.
Add 0.95g CsCl per ml. Invert gently until completely dissolved.

Into disposable ultracentrifuge tubes, pipet ethidium bromide. Wear gloves.
Approximately 200ul of 10mg/ml ethidium bromide per 10ml of CsCl/DNA solution.

Pipet the CsCl/DNA solution into the tubes. Use a pasteur pipet as a funnel.
Bring the volume up to the neck with a separate solution of CsCl.
This is made by adding 0.95g CsCl per ml to 25% sucrose, 50mM Tris.

Balance the tubes to within 0.01g. Seal the tubes.

Put the tubes in a clean,dry rotor.

If necessary, cover the tubes with metal caps that weigh the same.
Make sure the O-ring is seated properly.

Spin 50,000rpm at 15oC for about 36 hours.

Break down to 1000-800rpm, then let brake off.

Remove tubes carefully. Wear gloves.
Gently open the tubes at the top.
Insert a 21-20G needle in 3ml syringe just below lower band.
Pull lower band, remove needle carefully, put solution into polyallomer 5ml tube.
Empty tube into bleach. Discard needle in sharps bucket.

Extract solution at least twice with isopropanol over salt-saturated water.
Dialyze or precipitate the DNA. To precipitate, double the volume with TE;
add 2 volumes ethanol; ice, spin.

RT-PCR Protocol

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures

from

Jack Vanden Heuvel
Penn State University
Department of Veterinary Science
and Molecular Toxicology Program

Link

RT-PCR Protocol

Protocol for competitive RT-PCR

This protocols is from the lab of Dieter Kaufmann, Department of Human Genetics, University of Ulm, Germany

Link

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology
ISBN: 047150338X
Author: Roger Brent / Robert E. Kingston / J. G. Seidman / Kevin Struhl / Frederick M. Ausubel / Virginia Benson Chanda / David D. Moore / J.G. Seidman / F.M. Ausubel
Publisher: John Wiley & Sons Inc
Edition: ringbou edition (December 4, 2003)
ISBN: 047150338X
Ring-bound: 1600 pages
URL: /http://www.amazon.com/exec/obidos/redirect?tag=songstech-20&path=ASIN%2F047150338X
Summary:

Current Protocols in Molecular Biology, the first Current Protocols publication, remains the international standard by which all other lab manuals are judged. Basic methods for DNA preparation and isolation, library screening, and sequencing have been joined by more advanced procedures detailing DNA-protein interactions, yeast manipulation, and phosphorylation analyses. From basics to the cutting edge, CPMB is the only resource you need for successful experiments.

Link

SV40 Protocols-Ebook (Methods in Molecular Biology)

Book Properties
ISBN: 0896036537
Title: SV40 Protocols (Methods in Molecular Biology)
Author:
Publisher: Humana Press

Link

Standard PCR Protocol

Standard PCR Protocols

This protocol is link to Molecular Biology Techniques Manual
Third Edition
Edited by:
Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

Read Full Protocol

Overlap Extension PCR

Overlap Extension PCR is used to create long DNA fragments from short ones.

or used for Engineering the replication of target DNA through cloning, or changing its genetic code through mutations

PCR amplify the necessary fragments, using polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temp.
Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
Use proofreading enzyme for extension. Run 3 reactions of 10,15 and 30 PCR cycles without end primers. (Template extension step) Add end primers, then continue cycling for another 15-20 rounds. Gel extract the correct fragment. Clone into a your desired vector.

check out the latest Nature Methods Protocol
http://www.nature.com/nmeth/journal/v4/n5/pdf/nmeth0507-455.pdf

The Cell - A Molecular Approach-E Book, The Cell - A Molecular Approach Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000

 

 

 

 

The Cell - A Molecular Approach

Cooper, Geoffrey M.

Sunderland (MA): Sinauer Associates, Inc. ; c2000

Read This Book Full Version

Cancer Medicine- Ebook, Kufe, Donald W.; Pollock, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C., Jr.; Gansler, Ted S.; Holland

Kufe, Donald W.; Pollock, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C., Jr.; Gansler, Ted S.; Holland, James F.; Frei III, Emil, editors. Hamilton (Canada): BC Decker Inc. ; c2003

Read This book Full

TA cloning or Subcloning of PCR Products Protocol

TA Subcloning of PCR Products

This procedure is adapted from D. Marchuk, M. Drumm, A. Saulino, and F.S. Collins Nuc. Acids. Res. (1991) 19:1154.

CONSTRUCTION OF T-VECTOR

1. suspend 10ug pUC 19 in:
   • 4.0ul 10X reaction buffer (we use Bo. Mann. buffer A)
   • 2.0ul (20U) Sma I
   • X ul dwater to a total vol. of 40ul
     Incubate at 30 (not 37) degrees for 1 hour. This is easier if done in a 0.4ml tube in a thermal cycler.
2. Heat to 70 degrees for 15 min. to kill the enzyme
3. Bring to 100ul w/ water (add 60ul).
4. Extract w/ phenol, phenol/chloroform and then chloroform.
5. add 9ul 3M sodium acetate.
6. ppt. in ETOH, wash with 70% ETOH (be careful with the pellet!).
7. Dry in spin vac at room temp (do not use heater!).

********************T-TAILING THE VECTOR******************

At this point, it is assumed that there has been 80% recovery of the cut plasmid DNA.

1. Resuspend the plasmid DNA in 63ul water (conc approx. 130ng/ul)
2. To the resuspended plasmid add:
   • 10ul 10X PCR buffer (standard cetus stuff, no MgCl)
   • 20ul 10mM dTTP [2mM final]
   • 6ul 25mM MgCl2 [1.5mM final]
   • 1ul Taq polymerase (Cetus amplitaq 5U/ul)
   • ______
   • 100ul total volume.
3. Incubate for 3 hours at 70 degrees C.
4. Extract with Phenol, Phenol/chloroform, chloroform.
5. Extract twice with ether (so I'm paranoid!)
6. add 75ul 2M **ammonium** acetate (assuming 75ul recovery from extractions).
7. Add 150ul isopropanol. Spin 20mins in microfuge at full speed at 4 degrees.
8. Wash with 70% ETOH 9 Dry pellet in spin vac and store at -20 degrees until use.

TREATMENT THE PCR PRODUCTS

" If you can see it, you can clone it".

1. Add an equal volume of chloroform (*NO* IAA) to the PCR reaction and spin 1-2 minutes in microfuge at RT.
2. Remove the oil which is now on the ****BOTTOM***.
3. Spin again for two minutes and remove the last little bit of oil from the bottom. You will know when you have gotten it all when you see the interface in the pipette tip. It is important that all the oil be removed otherwise subsequent procedures will be very difficult.
4. Add 100ul 4M ammonium acetate, vortex, and then add 200ul isopropanol.
5. Centrifuge 20min at 4 degrees, wash in 70% ETOH.
6. Dry in speed vac.
7. Resuspend the DNA in 8-10ul TE, add loading buffer and load onto a 4% Nusieve (TAE) agarose gel. Run until the desired band is well separated. The more DNA in the band, the easier it is to subclone.
8. Cut out the band. Minimize the exposure of the gel (and you!) to short wave UV

LIGATION OF PCR PRODUCTS TO T-VECTOR

1. Heat the gel containing the PCR fragment to 65C for 10 minutes, place in a 37C water bath or block and add to a separate tube (also at 37C):
   • 10 ul gel
   • 4ul 5X ligase buffer (commercial buffer that comes with BRL T4 ligase)
   • 4ul water
   • 1ul vector (25-50ng)
   • 1ul ligase
   • Incubate at 12C overnight.
2. Heat the mixture to 68 degrees for 5 minutes and add 100ul water.
3. Extract with phenol, phenol/chloroform, and chloroform. These steps are to remove the agarose.
4. Add 10ul 3M NaAcetate and precipitate with ethanol.
5. Wash the pellet in 70% ETOH, dry in the speed-vac. Resuspend in 5ul of water just prior to transformation.

*Transformation - We usually use electroporation into XL-1 blue cells. You need cells that can achieve at least 1 x 10^7 transformants per ug of DNA if a CaCl based protocol is used.

*Storage: The T-vector should be stored at -20C at all times. When stored in dry form, the T-overhangs will last longer (I don't know how long yet). In solution, it lasts at least a couple of weeks at - 20C.

*Enzymes - The batch of SmaI that is used is particularly critical. Some are contaminated with an endonuclease that removes a few bases from the cloning site. The batch of smaI should be checked before it is used to cut vector for cloning purposes. If bluescript is used, EcoRV can be substituted for sma I.

txpljfg@uabcvsr.cvsr.uab.edu

Cloning of Blunt-end PCR Fragments Protocol

Direct Cloning of Blunt-end PCR Fragments

BioTechniques 13:613

• Phenol extract the PCR product
• Ethanol precipitate
• Treat for 1hr at 37C with 10 units of T4 DNA polynucleotide kinase and 10 units T4 DNA polymerase I(NEB) in a 100ul reaction volume containing 50mM Tris-HCl pH7.5, 10mM MgCl2, 1mM DTT, 50 ug/ml BSA, 1mM ATP, 200uM each dNTP.

I run the entire thing out on a 1.3% agarose TAE gel, cut a trough in front of the band, pour in some 0.7% LMP agarose(BRL), run the product into it and excise.

The PCR product in the LMP can be used for ligations directly, without purification. The ligations take place at room temp on the benchtop. I prepare the vector with minimal digestion (~2hr) then treat it with shrimp alkaline phosphatase(USB). I usually prepare a stock of this vector to have on hand, so I know it is good and will have a low background. You may also want to try using an EcoRV cut vector instead of a Sma cut vector.

• Remove the oil with Diethylether.
• To 40 ul of the PCR reaction add 50 ul of H2O. Add 10 U T4 PNK, 10 U klenow, ATP (to 1 mM) and some more dNTPs (usually 4 ul of 1.25 mM...whatever) and icubate at 37 oC for 30 min.
• Phenol/chloroform extract
• chloroform/IAA extrac
• EtOH precipitate

you can then just "shotgun" clone this DNA. However, if you have several non-specific bands then you may want to gel purify the fragment first.

One point to note....

We have given up blunt end cloning into Sma I sites where possible. Several people have reported problems with Sma I cut DNA. By choice we clone into EcoRV sites.

Agarose Gel Electrophoresis-Protocol

Agarose Gel Electrophoresis

Caution: wear gloves because of Etedium Bromide(Cancer Causing).

1. Dilute the 10X running buffer (TBE with EtBr) to 1X. Calculate the amount of the 1X buffer in gel tray(s). Add appropriate amount of agarous (for 5 mm standard gel; 0.7 % or 1 %) in the buffer, melt in the microwave oven. Pour the melting agarous (appr. 40-50 C) into a graduate cylinder, add 1X buffer up to the pre-determined volume, pour back into the flask to mix, and fill the gel tray (watch out bubble). Let set at least one hour to harden.

    Gel size (W X L) Agarous (Rec)  
    Gel tray   (cm X cm)  Owl  Rec. Buffer (L)  1.0 %   0.7 %
    
    A3    20 X 40   600  400  3.0   L 4.0   2.8 
   
    B2    12 X 14   130  100  0.5   L 1.0   0.7
   
    C1   7.6 X 5.1   30   20  0.1   L 0.3   2.7
    
   

   
   
2. Fill an electrophoresis chamber with 1X running buffer until the gel is covered by a couple of millimeters.
   
3. Adjust DNA concentration of sample (appr. 5 to 8 �g genomic DNA; 0.5 �g plasmid DNA per lane).
   
4. Load the DNA samples (with 1/10 volume of tracking dye and heat shocked for 5 min at 65) carefully with a Pipetman P20 by slowly expelling the solution into a well, with the pipet tip slightly below the top of the well. Do not hold the pipet too far in the well: the sample will sometimes come out the bottom. Try not to plug the pipet tip against the side of the well either: the sample will usually squirt out when there gets to be enough pressure from the pipetman.
   
5. Load the appropriate marker DNA flanking the sample lanes.
   
6. Close the lid on the electrophoresis kit. Always run the DNA toward the red (+) terminal. Electrophoresis the gel at 11 volts overnight or 100 volts for a couple of hours.
   

   Note: the size of DNA to be separated needs to be matched to the agarous concentration:

   Agarose %	Range of separation
      0.3		60 - 5 kb
      0.6		20 - 1 kb
      0.7		10 - 0.8 kb
      0.9		 7 - 0.5 kb
      1.2		 6 - 0.4 kb
      1.5		 4 - 0.2 kb
      2.0		 3 - 0.1 kb
   

Mapping our genes: the genome projects : how big, how fast?-Ebook

DIANE Publishing

ISBN 142892258X

Read this book online

What is DNA REPAIR?

simple again.. you damage ur DNA by smoking.. n repair it by urself...learn how..