PCR Protocol--Ligation Mediated Suppression PCR

Ligation Mediated Suppression PCR

(adapted from McKinney et al., 1995, Siebert, 1995, Strauss et al., 2001 and Alonso et al., 2003)

PURPOSE: To analyze unknown flanking genomic sequences adjacent to a T-DNA left border

1.) Isolation of DNA
-collect 2-3 young leaves in an eppendorf tube
-add 100 uL extraction buffer and add proteinase K, grind tissue using a blue pestel (no large pieces of leaf should be left).
-add another 100 uL of extraction buffer, vortex, and incubate in 37C for 30 min.
-add 200 uL of saturated phenol and vortex.
-spin at max speed in centrifuge for 2 min.
-collect upper phase to new eppendorf tube.
-add 200 uL of (24:1) chloroform:isoamyl alcohol, vortex, centrifuge at max speed for 2 min.
-collect upper phase into a new eppendorf tube.
-add 18 uL of 3M sodium acetate and add 400 uL of 100% EtOH, mix by inverting and incubate for 10 min at 4C.
-spin in centrifuge at max speed for 10 min.
-pour supernatant off and wash with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour supernatant off and wash again with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour off supernatant
-carefully pipette off excess EtOH
-let pellet dry for 45 min in the hood.
-resuspend DNA in 100 uL of TE
-store in -20C


2.) Digestion
-mix together:
50 uL gDNA (from above)
10 uL 10x Buffer 2
1 uL Hind III
39 uL dH2O
TOTAL volume 100 uL

-digest overnight at 37C


3.) Digestion Clean Up
-heat inactivate at 65C for 20 min.
-add 100 µL of chloroform
-mix by inverting tubes
-spin in centrifuge at max speed for 5 min.
-collect upper phase into a new eppendorf tube with 200 uL of isopropanol
-mix by inverting tubes
-incubate at room temperature for 10 min
-spin at max speed for 10 min
-remove supernatant
-wash with 100 uL of 70% EtOH
-spin at max speed for 5 min
-remove supernatant
-dry in hood for 45 min.
-resuspend in 20 uL of dH2O


4.) Constructing adapters for ligation
*adapters for ligation to Hind III ends are made by annealing oligos ADAPS-E1(5’-aattcacctgcccgg/3AmMc7/-3’) w/ a 3’ amino terminal end and ADAPL-E1(5’-ctaatacgactcactatagggctcgagcggccgcccgggcaggtg-3’). Oligos may be purchased from IDT @ www.idtdna.com

-dilute ADAPS and ADAPL to 100uM
-combine in equal amts of ADAPS and ADAPL (i.e. add 10 µL of ADAPS add 10 uL of ADAPL)
-vortex briefly
-place tube in 500 mL of boiling H2O for 2 min
-remove heat and let bath cool for 1 hr. (this is to ensure correct nucleotide pairing)
-store adapter at -20C


5.) Construction of Adapter Library (ligate adapter to digestion)
-mix together:
10 uL cleaned gDNA digestion
1 uL Adapter (100uM)
2 uL T4 ligase (NEB product)
2 uL 10 x T4 ligase buffer (NEB buffer)
5 uL dH2O
TOTAL volume 20 uL
-vortex and incubate at 16C overnight in thermocycler
-heat inactivate at 65 for 20 min
-add 180 uL of TE (this is your adapter library store at -20C)



6.) Primers for 1º and 2º PCR

*Primary products are generated from amplifying primers AP1 (5’-ggatcctaatacgactcactataggc-3’) and PgwLat52LB-WP1 (5’-ctatgttactagatcgaccgg-3’).

*Secondary products are generated by diluting primary products by 50 fold and amplifying with primers AP2 (5’-tatagggctcgagcggccg-3’) and PgwLat52LB-WP2 (5’-caattcggcgttaattcagtac-3’).

-Primers come from IDT and must be diluted to 10 uM concentration before using.



7.) Primary PCR

-mix together:
0.125 uL Ex Taq (Takara)
2.5 uL 10 x Ex Taq Buffer
2.0 uL dNTP Mix
1.0 uL AP1
1.0 uL WP1
17.375 uL dH20
1.0 uL Adapter Library
TOTAL volume 25 µL

*Run on LMS_PCR2 (conditions recommended by Takara)
1.) incubate at 94C for 2 min
2.) incubate at 94C for 30 sec
3.) incubate at 55C for 30 sec
4.) incubate at 72C for 1 min
5.) recycle to step 2 for 29 more times
6.) incubate at 65C for 10 min
7.) hold at 4C forever


8.) Secondary PCR
*Dilute primary PCR:
-98 uL of dH20
-2 uL of primary PCR

*Rxn is setup exactly like primary PCR.


9.) Run on 1% Agarose Gel
5g Agarose Low
500mL 1 x TAE
50uL EtBr

*Always run a ladder to verify our bands are between 200 and 2000bp. Should only sequence the lines that give a clear band.


10.)Sequencing
*Need to clean up PCR rxn using Qiagen or Eppendorf kits. Then setup the rxn to be sequenced.

- mix together:
2 uL cleaned secondary PCR
1 uL secondary primer (WP2)
17 uL dH20
TOTAL volume 20 uL

*Sequencing is done through UNC Lineberger Comprehensive Cancer Center (located on campus), and normal turn around time can range from 1-7days. To access sequences go to http://152.19.68.152/gafsite/Main.asp (listed as UNC-CH Genome Analysis under favorites).

Run sequences through Signal website http://signal.salk.edu/cgi-bin/tdnaexpress.

Unmapped lines can either be digested with a different enzyme (EcoRI),can try sequencing using PgwLat52RB primers, or try TAIL PCR.
*If you use EcoRI you must use the EcoRI adapter and then proceed as normal


Special Notes:
After secondary (nested) PCR, only lines with clear bands are sequenced. Bands are typically between 200 and 2000bp. Lines resulting in a smear or no bands will not provide good sequence. Not all lines with distinct bands will provide good sequence either (some bands are likely artifacts).

In our hands, LB mapping of EcoRI digests has up to 75% success rate. The remaining lines can be mapped by a combination of RB-mapping (with appropriate RB primers), digesting with a different enzyme (i.e. HindIII) (with appropriate Adapter modifications), or TAIL PCR.

The WP1 and WP2 primers described above are specific to pBI121 and vectors with pBI121 left border regions. Similar primers can be designed to sequence from right borders. The nested primer (WP2) should be ~50 bp inside the T-DNA region to accommodate the possibility of border truncation.

Multiple inserts complicate the results. It is possible that each independent insert could result in a distinct nested PCR band. Bands can be excised and sequenced. It is possible to get sequence from both bands, however, by directly sequencing the nested PCR products.


Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657

Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23: 1087-1088

PCR Protocol--Differential Display PCR

Differential Display PCR

About Differential Display PCR: All living organisms have thousands to tens of thousands of unique genes encoded in their genome, of which only a small fraction, perhaps 15%, are expressed in any individual cell. Therefore, it is the temporal and spatial regulation in gene expression that determines life processes. The course of normal cellular development as well as pathological changes that arise in diseases such as cancer are all believed to be driven by changes in gene expression. A pressing problem is to identify and characterize those genes that are differentially expressed in order to understand the molecular nature of disease state and subsequently, to devise rational therapies. Differential Display was invented in 1992 by Drs. Arthur Pardee and Peng Liang to allow rapid, accurate and sensitive detection of altered gene expression (Science. 1992, 257:967; U.S. Patent 5,262,311).

Further readings about differential display technique
What's Differential Display (GenHunter)Introduction to differential display technique
Differential Display (Chun-Ming Liu)The following procedures are described:
RNA extraction and qualification
DNase treatment of RNA sample
Reverse transcription
PCR amplification
Separation on acrylamide gels
DNA extraction from bands of interest
Re-amplification by PCR
Separation on agarose gels
Excision of amplified products
Differential Display (Breeden Lab)Detailed protocol for differential display
Differential Display (Plant Molecular Biolgy Lab)It's for plant RNA display. The protocol should be general.

Differential Display-Reverse Transcription-PCR (Gerard Lazo)

Rational primer design greatly improves differential display-PCR.

Applications of Differential-Display Reverse Transcription-PCR.

Differential Display-PCR -- Sambrook and Russell.

Development and optimization of a fluorescent differential display.

The Science Advisory Board – Differential Display PCR.

Perspective: Micoarrays and Differential Display PCR.

Laboratory Techniques: Differential Display - Polymerase Chain Reaction
Effect of Primer Purity on the Banding Patterns of DifferentialDisplay Polymerase Chain Reaction
Differential Display of RNAs (Breeden Lab.)

PCR Protocol--Colony PCR Protocol

Colony PCR Protocols

Colony PCR protocol
Detailed protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/colony_pcr.html
Colony PCRColony PCR. This protocol is designed to quickly screen for plasmid inserts directly from E. coli. colonies. The plasmid should be high copy number such as ...www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf
Colony PCR ProtocolColony PCR Protocol. 1. Pull out eight glycerol stock plates from the –80. o. C freezer and set on bench top to. thaw. Be sure to remove the foil seal ...microarrays.berkeley.edu/file_download.php?fid=13
Colony PCR ProtocolColony PCR Protocol. Overnight Culture 5.0µl. 10x PCR buffer 5.0µl. MgCl2 (25mM) 5.0µl. Forward primer (10µM) 0.2µl. Reverse primer (10µM) 0.2µl ...www.unc.edu/~fconlon/Protocols/Colony%20PCR%20Protocol.doc
Colony PCR
Colony PCR Protocol contributed by Lynn Hancock 1. Choose a reasonable sized colony (2-3 mm in diameter) and resuspend 100 µl ddH2O. 2. For PCR, ...www.enterococcus.ouhsc.edu/ColonyPCRProtocol.asp
Yeast Colony PCR -- Amberg et al.
Search CSH Protocols. Advanced Search · Find Protocols · Find a Kit · Access Personal Page · Submit Protocols · Help · Subscribe · About CSH Protocols ...www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4170
Colony PCR - OpenWetWare
From OpenWetWare. Jump to: navigation, search. See Colony PCR for general information about this protocol and other variants ...openwetware.org/wiki/Endy:Colony_PCR
Colony PCR protocol
Monserate Biotechnology Group Custom services, Colony PCR protocol.www.monseratebiotech.com/colony-pcr.html
Yeast Colony PCR protocolYeast Colony PCR protocol. derived from http://sequence-. www.stanford.edu/group/yeast_deletion_project/deletions3.html.
Colony PCR (Forsburg Lab)
The Forsburg lab pages: fission yeast colony PCR protocol. ... Colony PCR can be used to identify colonies where your favorite gene yfg1 has been replaced ...http://www-rcf.usc.edu/~forsburg/pcr.html
Rapid Screening by Direct Colony PCR Using the FastStart PCR Master
The FastStart PCR Master is a convenient and ideal tool for direct colony PCR. Intact bacteria can be analyzed directly without prior template purification, ...www.analytica-world.com/articles/e/65346/

PCR Protocol--Asymmetric PCR

Asymmetric PCR

What is asymmetric PCR? A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.

Asymmetric PCR Protocol 1. Pick a phage plaque and place in 100 ul TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 ul of TE/TX100 in a microcentrifuge tube. 2. Heat the tube for 10 min at 95C. 3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant. 4. Add the following components in a PCR tube: 5 ul of phage or bacterial extract (from Step #A3) 50 uM of dNTPs 50 pmol of Primer 1 1 pmol of Primer 2 in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 ul 2.5 Units of Taq polymerase 5. Run 30 to 35 cycles in a thermocycler using the following PCR program (see Hint #1 and #2) 95C for 60 sec 60C for 30 sec 72C for 2 min 6. Run a small aliquot on an agarose gel to analyze for single-stranded DNA (see Protocol on Agarose Gel Electrophoresis of DNA). 7. Purify the PCR products and sequence, if desired.
Asymmetric PCR for ssDNA ProductionHOT ASYMMETRIC PCR (Mullins Lab)
Direct sequencing by thermal asymmetric PCR

Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.

Detection of asymmetric PCR products in homogeneoussolution by fluorescence correlation spectroscopy
Asymmetric PCR Using the Primers Anchored on the surface of magnetic nanoparticles.

PCR Protocol--Long PCR Protocol

Long PCR Protocol

Kyle’s protocol for long PCR
Kyle’s protocol for long PCR. This protocol intended for generation of long PCR products (3-10kb) from mouse ES cell or tail DNA. General Considerations: ...www.whitelabs.org/Lab%20Protocols/PCR%20Protocols/kyle.htm
Long PCR Protocol
Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School, Boston, MA, USA.arep.med.harvard.edu/labgc/estep/longPCR_protocol.html
Tips for Long and Accurate PCR Tips for Long and Accurate PCR. Wayne M. Barnes. Department of Biochemistry and Molecular Biophysics, Washington University School of ...http://www.klentaq.com/products/lapcrchapter.pdf
Mullins Lab : Protocols : Long PCR
Two long PCR steps:. First round of the nested PCR step of the end point dilution procedure to quantitate the cDNA. First round of the nested PCR step of ...ubik.microbiol.washington.edu/protocols/other/longpcr.htm
Long PCR
LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION. Introduction. Long range PCR allows the amplification of PCR products, which are much ...europium.csc.mrc.ac.uk/WebPages/Database/Methods/longpcr.htm
Extra-Long PCR Ki, EPICENTRE Biotechnologies
EPICENTRE develops and manufactures reagents and kits for genomics, proteomics, and RNA research. EPICENTRE's products are used worldwide in a broad range ...www.epibio.com/item.asp?ID=306
Long PCR Protocol
Efficient Long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading ...wheat.pw.usda.gov/~lazo/methods/lazo/longpcr.html
Long PCR.
Long PCR. Cheng S, Chang SY, Gravitt P, Respess R. Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, California 94501. Mesh Terms: ...www.ncbi.nlm.nih.gov/pubmed/8208299
Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products.
Here we describe a practical procedure for sequencing long PCR products. The method relies on ultrasonic shearing of PCR products, resulting in fragments ...www.nature.com/nprot/journal/v2/n2/abs/nprot.2006.453.html
Long PCR Protocol
Aug 10, 2003 ... Introduction · Research Pages · People involved Photo galleries Publications Other sample sites Education · Links to other micobial ...www.hawaii.edu/microbiology/MO/longpcr.htm
Long PCR -- Sambrook and Russell.
Primers used for long PCR are generally slightly longer (25-30 nucleotides) than those used for standard PCR. It is particularly important to strive for ...www.cshprotocols.org/cgi/content/full/2006/2/pdb.prot3841
Efficient Cloning of Long PCR Inserts with the In-Fusion PCR Cloning System.Clontech’s In-Fusion PCR cloning method. provides superior results when applied to the. challenging task of cloning long PCR fragments. ...www.clontech.com/images/ctq/APR07UPD/CR742319_LongPCR_IF_US.pdf
Nature Protocols: Long PCR amplification of the entire mitochondrial genome from individual helminths for direct sequencing.
In this article, we describe a practical long PCR approach for the amplification and subsequent sequencing of the entire mt genome from individual helminths ...www.natureprotocols.com/2007/09/20/long_pcr_amplification_of_the.php
Long PCR - MyBio
Long PCR Reagents and GuidelinesLong PCR Reagents and Guidelines. ... General Guidelines for Long PCR Conditions and Enzyme Mixtures Efficient Long PCR ...mybio.wikia.com/wiki/Long_PCR

PCR Protocol--Methylation Specific PCR

Methylation-specific PCR: a novel PCR assay for methylation status ...
Selected References. Page Browse · PDF (1.9M) · Contents · Archive. Related material:. PubMed record, PubMed related arts, PubMed LinkOut, Substance ...www.pubmedcentral.nih.gov/articlerender.fcgi?artid=38513

PCR Protocol--PCR Application Manual

PCR Applications Manual (from Roche Diagnostics).