IGEM:MIT/2006/Notebook/2006-8-3

Today's big plan

 * 1) design pchBA primers -done
 * 2) *pchB forward
 * 3) *pchA reverse
 * 4) *2 pchA mutagenesis
 * 5) PCR cleanup BAT2 and THI3 [nanodrop] -done
 * 6) miniprep overnight LCs [nanodrop] -done
 * 7) LC R0040-E0840 -done
 * 8) start digests -done: 11:30
 * 9) sequence correct (3) ATF1mut-Term assemblies -done
 * 10) *discard all ATF1mut-Term plates b/c we have an index plate -done
 * 11) autoclave waste -done
 * 12) pour plates (A/T, A) -done
 * 13) design gels (small wells for 3-part assemblies versus big wells for gel extracts) -done
 * 14) digest gel expectations????? -done
 * 15) heat shock digests -done
 * 16) load 3 digest product gels (and run THI3 again to get more DNA) -done
 * 17) PCR clean-ups on necessary digests [nanodrop] -done
 * 18) read digest gels and design ligations based on results -done
 * 19) gel extraction -done
 * 20) ligate (15 mins) -done
 * 21) design TH13 mutagenesis primers -done
 * 22) check all primers and email order to Heather -done
 * 23) transform -done
 * 24) label plates -done
 * 25) set a team meeting time -done

Template Idea
../../Template

Primer To Do

 * 1) Order new pchBA primers

Miniprep/Digest To Do

 * 1) Miniprep BSMT.B0015 LC
 * 2) Cut BSMT.B0015 with XP
 * 3) Miniprep B0030
 * 4) Cut B0030 with ES and SP(bkb)
 * 5) Miniprep R0040
 * 6) Cut R0040 with ES and SP(bkb)

WG Ligations/Transformations To Do

 * 1) Gel extract BSMT.B0015XP
 * 2) PCR Cleanup B0030SP backbone
 * 3) Ligate BSMT.B0015XP and B0030SP backbone-> transform
 * 1) Ligate B0030ES with BSMT.B0015XP in A/CEP
 * 2) Ligate B0030ES with BSMT.B0015XP in A/TEP

osmY & E0840 Ligations/Transformations To Do

 * 1) osmY and A/CEP and A/TEP
 * 2) osmY cut with SpeI
 * 3) E0840 cut with XP
 * 4) Ligation of osmY cut with S and E0840 cut with XP and pSB1AK3 cut with EP

ATF1 Miniprep/Digest To Do

 * 1) Miniprep ATF1.B0015 LC
 * 2) Cut ATF1.B0015 with XP
 * 3) Gel extract some of ATF1.B0015XP

ATF1 Ligation/Transformation To Do

 * 1) Ligate ATF1.B0015XP with B0030ES in A/CEP and A/TESP

IAA Digest

 * 1) BAT2 with EP, ligate with A/C and A/TEP
 * 2) THI3 with SX
 * 3) pSB1AK3 with SX

IAA Ligations

 * 1) BAT2EP w/ A/C & A/T BKB
 * 2) THI3SX w/ psBIAK3SX->A/K plate

new pchBA primers
Primer pair 1 *   Forward: 5' CCGCTTCTAGATGAACTCTAGCTATACACAG 3' Reverse: 5' CTGTGTATAGCTAGAGTTCATCTAGAAGCGG 3' *    GC content: 45.16%           Location: 40-70 Melting temp: 75.2°C        Mismatched bases: 1 Length: 31 bp               Mutation: Substitution 5' flanking region: 15 bp   Forward primer MW: 9439.27 Da     3' flanking region: 15 bp    Reverse primer MW: 9590.34 Da
 * pchA reverse new: G TTT CTT TAC TAG TAT TAT Tag gcg acg ccg cgc tg [melt temp = 65.6, no hairpin]
 * pchB forward new: GTT TCT ACG AAT TCG CGG CCG CTT CTA Gat gaa aac tcc cga aga ctg cac
 * pchA mut forward new: gat cga ccc att gga ccc gct aca ggt att cgg tgc
 * pchA mut reverse new: gca ccg aat acc tgt agc ggg tcc aat ggg tcg atc
 * THI3 mut: possible problem...5 base pairs match up to create hairpin....

Primer pair 2 *   Forward: 5' GCCGCTTCTAGATGAACTCTAGCTATACACAG 3' Reverse: 5' CTGTGTATAGCTAGAGTTCATCTAGAAGCGGC 3' *    GC content: 46.88%           Location: 39-70 Melting temp: 76.7°C        Mismatched bases: 1 Length: 32 bp               Mutation: Substitution 5' flanking region: 16 bp   Forward primer MW: 9768.48 Da     3' flanking region: 15 bp    Reverse primer MW: 9879.53 Da