TITLE: Genomic mapping of transformed DNA fragments
AUTHORS: Oskar Johansson, Adrian Clarke

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mapping of transformed DNA within mutated diatoms. A stepwise specific PCR reaction that allow for identification of genome location of transformed fragments such as antibiotic resistance genes or GFP constructs. </div><div class = "text-block">Degenerate primer 1:     5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCTNASATANSATANT TC - 3'</div><div class = "text-block">Degenerate primer 2:     5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCCNTSABGNACYTN CTG - 3'</div><div class = "text-block">Degenerate primer 3:     5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCNGACGARWGANA WGAC - 3'</div><div class = "text-block">Degenerate primer 4:     5' - GCCGGACTTAGCGAGCA TGTGTAGTACCTTGCCG ATCCTAHATGDAGKACN TAC  - 3'</div><div class = "text-block">Specific primer 1:     5' - GCCGGACTTAGCGAGCA TGTGTAG - 3'</div><div class = "text-block">Specific primer 2:     5' - CATGTGTAGTACCTTGC CGATCC - 3'</div><div class = "text-block">Internal primers must be designed with regard to each construct that are to  be mapped. They should not overlap but instead cover consecutive stretches of known DNA sequence of the transformed construct. </div></div>

[STEPS]
?. Primary, 25uL PCR reactionVol.         Component5uL         5x SuperFi Buffer0.8uL     dNTP - Mix (10mM Each)3uL         Internal Primer (10uL)0.2uL     SuperFi12.5uL   Water2.5uL     Degenerate Primer (40uM)1uL        gDNA (100ng/uL)Primary PCR programStep    Temp     Time1             96         5 min2             98         10 sec3             70         15 sec4             72         3 min5    Goto 2 - 30 more times 6             98         10 sec7             25         1 min8             Ramp to 72 0.5C per sec 9             72         3 min10    Goto 6 - 1 more time 11             98         10 sec12             70         15 sec13             72         3 min14             98         10 sec15             70         15 sec16             72         3 min17             98         10 sec18             70         15 sec19             72         3 min20             98         10 sec21             40         45 sec22             72         3 min23    Goto 11 - 10 More times 24             72         5min25             4            Forever
?. Dilute Primary PCR reaction 40x times (5uL + 195uL water)
?. Secondary, 25uL PCR reactionVol.        Component5uL        5x SuperFi Buffer0.5uL     dNTP - Mix (10mM Each)1.5uL     Internal Primer 2 (10uM)0.2uL     SuperFi1.5uL     Specific primer 1 (10uM)15.3uL   Water1uL         Diluted primary PCR reactionSecondary PCR programStep    Temp    Time1             98         30 sec2             98         10 sec3             70         15 sec4             72         3 min5             98         10 sec6             70         15 sec7             72         3 min8             98         10 sec9             57         30 sec10           72         3 min11           Goto 2 - 12 More times 12           72         5min13             4         Forever
?. Dilute Secondary PCR reaction 40x times (5uL + 195uL water)
?. Tertiary, 50uL PCR reaction Vol.       Component10uL     5x SuperFi Buffer0.8uL    dNTP - Mix (10mM Each)2uL       Internal Primer 3 (10uM)0.3uL    SuperFi2uL       Specific primer 2 (10uM)25.4uL Water2uL Diluted Secondary reactionTertiary PCR programStep    Temp    Time1             98         30 sec2             98         10 sec3             57         30 sec4             72         3 min5     Goto 2 - 14 More times 6             72         5min7               4          Forever
?. Run a 1% agarose gel, excise bands and cleanup using a gel cleanup kit (Ex. Promega Wizard)
?. Clone 2uL of purified bands into Blunt TOPO vector (Thermo Fisher Scientific) and transform competent E.coli dH5a (Thermo Fisher Scientific) by 30 sec heat shock at 42C. Grow 5mL overnight cultures of picked colonies and extract vectors.
?. Sanger sequence using the TOPO vectors internal primers.