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.

Large Scale Plasmid Midi Prep DNA Fingerprinting

Abstract:

The isolation of large amounts of plasmid DNA from insert-containing clones is necessary because subsequent DNA sequence analysis requires high levels of pure starting DNA. We will be using a moderately expensive plasmid isolation kit (Promega, Inc.) because it produces DNA compatible with our sequencing apparatus.  As compared with the rapid mini-prep, large-scale plasmid isolation methods all share three basic parts: (1) a way to gently lyse the cellular hosts; (2) a way to crudely separate plasmid from the total cell extract; and (3) a way to purify and concentrate plasmid DNA.

In order to compare your samples using DNA fingerprinting methods - you will set up restriction digests based on some moderately tricky pre-lab analysis. Recall that restriction enzyme digest reactions require template DNA, enzyme(s), an appropriate enzyme buffer, and some water. Today, you will set up uncut samples, single digests, and double digests using interesting enzymes that should reveal more complex cutting patterns than those produced by EcoRI last time. As you will see, there are many challenges to this. For example, two enzymes we want to cut at the same time may function to lesser extents in non-ideal buffers.  Charts are available in the form of company literature (in our case: Gibco BRL Life Technologies) to assist us when faced with the problem of making recipes and figuring these kinds of compatibility issues out. Additionally, you will analyze the enzyme's recognition sequence and calculate - based on probability - how many times each enzyme is predicted to cut the clone. Does the pattern you obtain match this prediction - why or why not?

See full Plasmid Isolation Procedures at

National Science Foundation

Western Oregon University

Yellowstone National Park

http://www.wou.edu/~boomers/Molecular/midi.htm

RT PCR Protocols and Analysis

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

Maria Marone^1*, Simona Mozzetti¹, Daniela De Ritis¹, Luca Pierelli¹ and Giovanni Scambia¹

¹ Department of Gynecology and Department of Hematology. Catholic University, L.go A. Gemelli 8, 00168 Rome. Italy.
* To whom correspondence should be addressed: Maria Marone, Department of Gynecology and Department of Hematology. Catholic University, L.go A. Gemelli 8, 00168 Rome. Italy. Email: maria.marone@tiscalinet.it

Link

http://www.biologicalprocedures.com/bpo/arts/1/20/m20.htm

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

RT-PCR Protocols ebook

This RT-PCR protocol book describes the detail of novel, useful, and
interesting RT-PCR applications.

protocols available for

highly sensitive detection and quantification of gene expression, the in situ localization of gene,
expression in tissue, and the cloning of genes,

as well as for analyzing T-cell clones and the differential expression of genes,

including laser-capture microdissection (LCM),

real-time and quantitative PCR,

microarray technology, cDNA cloning.

Book Info:
Published in 2002
Published by Humana press
Author Joseph OConnell
ISBN 0896038750  Size 4.30MB

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

Protocol for Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation

DNA Plasmid is isolated from 1-2 ml bacterial cultures by alkali and SDS treatment.

Link

SV40 Protocols-Ebook (Methods in Molecular Biology)

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

Link

PCR Protocols-Methods in Molecular Biology EBook

Publisher: Humana Press

Number Of Pages: 392

Publication Date: 1993-01

Sales Rank: 914567

ISBN / ASIN: 0896032442

EAN: 9780896032446

Binding: Spiral-bound

Manufacturer: Humana Press

Studio: Humana Press

Link

Pass: econiches

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

Primer Sets and PCR Manual

This 24-page brochure details each step of the Polymerase Chain Reaction process with technical information for basic PCR techniques, methods, applications and polymerase choices. You will want to keep this new booklet close at hand because it also includes FAQs, references and a troubleshooting guide.

Download

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

C. elegans-Ebook, Riddle, Donald L.; Blumenthal, Thomas; Meyer, Barbara

C. elegans II

Riddle, Donald L.; Blumenthal, Thomas; Meyer, Barbara J.; Priess, James R., editors.

Plainview (NY): Cold Spring Harbor Laboratory Press ; c1997

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
   

X-Gal Protocol (Promega Biotech)

X-Gal Protocol

Caution: X-gal stock solution contains Dimethylformide

X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) turns blue when incubated in the presence of b-glactosidase. This gene is on several of the cloning plasmids (especially, on the pUC series and lGT11 vectors). When an inserted piece of DNA is placed in the correct restriction site, the lacZ gene is interrupted and the colony does not turn the media blue (colony we want). Be sure to run controls.

1. Make 13X100 tubes with 2.5 ml LB and 0.8 agar by melting then dispensing it into the tubes and autoclaving it for only 5 min.
2. Use the tubes while hot or re-melt briefly and hold at 42 C.
3. Add 20 �l 20 mg/ml IPTG (filter sterilized in H2O), 50 �l 20 mg/ml X-gal (in Dimethylformide), and 1 �l Crb solution (100 mg/ml).
   Note: Dimethylformide seems to melt plastic so make the stock in glass or PP (or PA) oakridge tubes.
4. Add the transformed cells (try to get about 200 CFU in up to 250 �l). Vortex and overlay on a CA plate containing the appropriate antibiotic (usually Crb).
   * 2.5 ml is a little tricky to overlay neatly: do not replace cap, tilt the plate to get uniform coverage of the overlay.
5. Let the agar solidify then incubate at 37 C until the blue color develops.
6. Pick the colorless colonies to media with the correct antibiotic and verify the insert by mini plasmid preparations or colony hybridization.

How to Prepare of Competent Cells for transformation? (protocol)

Preparation of Competent Cells (from PMB)

1. Inoculate a couple of 20 ml LB broth in a 125 ml flasks with 0.5 ml of an overnight culture of the recipient strain.
2. Shake at 37 C until O.D. 600=0.13 - 0.15 (1 - 2 hours). Measure by taking some of the broth out with a sterile DPTP and reading in a plastic cuvette (discard cells afterward): If flanking arm flasks are accessible, use them for measuring O.D.
3. Spin the cells in sterile Oak Ridge Tube (5 K, 5 min, 4 C) and resuspend in 1 ml PMB #A. Then add another 9 ml PMB #A and spin the cells again (5 K, 5 min, 4 C).
4. Decant and resuspend in 1 ml PMB #B. Then add 9 ml more PMB #B.
5. Let sit on ice for 30 min, spin as above and resuspend in 2 ml PMB #B, add 0.2 ml glycerol and 30 �l DMSO. Use fresh or freeze in dry ice/EtOH.

PMB #A
1X 200 ml 1X
10 mM MOPS pH 7.0 210 mg Free acid
230 mg Na salt
10 mM Rubidium chloride 240 mg
Autoclave and store at 4 C
PMB #B
1X 200 ml 1X
10 mM MOPS pH 6.5 350 mg Free acid
85 mg Na salt
50 mM Calcium chloride 1.12 g
10 mM Rubidium chloride 240 mg
Autoclave and store at 4 C