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

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.B0015_XP
2. PCR Cleanup B0030_SP backbone
3. Ligate BSMT.B0015_XP and B0030_SP backbone-> transform

1. Ligate B0030_ES with BSMT.B0015_XP in A/C_EP
2. Ligate B0030_ES with BSMT.B0015_XP in A/T_EP

osmY & E0840 Ligations/Transformations To Do

1. osmY and A/C_EP and A/T_EP
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.B0015_XP

ATF1 Ligation/Transformation To Do

1. Ligate ATF1.B0015_XP with B0030_ES in A/C_EP and A/T_ESP

IAA Digest

1. BAT2 with EP, ligate with A/C and A/T_EP
2. THI3 with SX
3. pSB1AK3 with SX

IAA Ligations

1. BAT2_EP w/ A/C & A/T BKB
2. THI3_SX w/ psBIAK3_SX->A/K plate

new pchBA primers

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