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
