Populational Analysis of Genomic DNA Protocol

Article Details

Protocols » Article Details

Populational Analysis of Genomic DNA Protocol

Date Added: February 26, 2008 02:59:16 PM

Author: plinkadmin

Category: Genetics and Genomics

Populational Analysis of genomic DNA Amount of DNA depends on quality < 0.5 ug if good 2 ug if degraded. Use 1ug routinely. 0.25 uM of primers 0.5 mM dNTPS 1X #3 Buffer 0.1ul of enzyme for every 10 ul of reaction. (i.e 0.01ul E/ul) Typical Reaction 25 ul volume 1X #3 Buffer: 2.5 ul 10mM dNTPs: 1.25 ul Primer 4 and 3* [2.5uM@]: 2.5 Enzyme Expand BMC: 0.25 ul dH2O: 16.5 ul TOTAL --23.0ul-- 2ul of DNA 80�C 10-30 s, 5X {93�C, 20s; 56�C, 20s; 68�C, 7min}, then: 27X {93�C, 20s; 62�C, 20s; 68�C, 7min+ 20s/cycl} *************************************************** -Approximately (very appr) 0.05 to 0.2 g of tissue (spleen, liver) Sonicate in 350 ul of SET (1% SDS, 50mM EDTA, 100mM Tris, pH 7.8) -heat to 80-85�C 10 min, cool to 68�CAt 68�C -add 20 ug of Proteinase K, incubate 1- 2hours -After protease digest 5 organic extractions: 3 phenols, 1 phenol/CIA, 1 CIA. 1st phenol should be hot, and mix well (1-2 minutes) -Ethanol precipitateBring up in 200ul of 0.1x T.E. PCR with 50-75 (FINAL 0.1 TO 0.5 uM 11/95) pmol or primers, 0.2 to 1ug of DNA 25ul/reaction Ingredient: 1X, 10X 10X Taq buffer: 4 ul, 40 ul 25mM MgCl2 (2mM final): 3.2 ul, 32 ul 2mM dNTPs: 4 ul, 40 ul Taq enzyme (Promega): 0.5 ul, 5 ul dH2O: 13.3 ul, 133 ul Total: 25ul, 250 ul In PCR tube 15 ul dH2O+DNA+primers (25ul/rxn) 1 ul DNA (0.5 ug) 1 ul primer @ 60 pmol/ul 12ul dH2O heat 98�C 2 min, quick cool add master mix TouchDown (PCR cycle) 94�C 40s---74�C 40s -0.5�C@cycle 20cycles, 62�C 15 cycles +1s/cycle Run on 2+1% gel (regular + NuSieve) for <400bp ******************************************* IF A SINGLE BAND from PCR (if not gel purify) KILL Taq, 99�C 10 min, Slow cool to 37�C 30-45 mins. Digest with Xho and KpnI Add 50 of 1X #1 buffer, 1 ul of @ enzymes Ingredient: 1X, 10X dH2O: 43 ul, 430 ul 10x #1 buffer: 5 ul, 50 ul Xho enz.: 1 ul, 5 ul Kpn1 enz.: 1 ul, 5 ul digest 3-12 hours. 50ul/rxn Wizard prep or gene clean DNA to elimante small pieces and primers. Elute into 50 ul dH2O ligate 10 ul of PCR product with 2 ng of digest KS II Xho/Kpn Ingredient: 1X, 10X PCR product in dH2O: 12.5 ul fresh 10X ligation buffer: 1.5 ul, 15 ul T4-Ligase: 1 ul, 10 ul KSII plasmid(10ng/ul): 0.2 ul, 2 ul Total: 2.5ul/rxn 16�C 4-12 hours

Ratings You must be logged in to leave a rating.
Average rating: (0 votes)

Comments