IGEM:MIT/2005/Inventory
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File:PlasmidInventory IGEM2005.xls
Strains
- NB115 (pCT-4-4-20) -- 7.02 -80°C
- NB116 (pCT302 S101A) -- 7.02 -80°C
- NB118 (pCT-4M5.3) -- 7.02 -80°C
- MC4100
- Genotype: F- araD139 delta(argF-lac)U169 prsL150 relA1 deoC1 rbsR fthD5301 fruA25 lambda- [s]
Plasmids
- File:PCT302.pdf
- [[../PCT302 (aka pCT-4-4-20)/]]-- 7.02 4°C
- [[../PCT302 S101A/]] -- 7.02 4°C
- [[../PCT-4M5.3/]] -- 7.02 4°C
PCR Amplification
- Spin PCR purification kit ()
- Platinum, TAQ polymerase (DNA polymerase for PCR)
- DNTP mix (25mm ea. dntp) (nucleotides for PCR)
Agarose gels
- Agarose 100g (pouring gels)
- 10X TAE buffer (pouring/running gels)
- 1 kb DNA ladder 250µg (molecular weight standard)
- Ethidium Bromide Solution (10mg/mL) (staining DNA)
Cutting/joining DNA fragments
- EcoR1 10,000 u (Restriction Enzyme)
- PstI 10,000 u (Restriction Enzyme)
- SpeI 2500 u (Restriction Enzyme)
- Xba1 3,000 u (Restriction Enzyme)
- T4 DNA Ligase 100,000un (Ligating DNA fragments)
Purifying DNA
- Spin Plasmid Kit, Mini(for minipreps)
- Gel Extraction Kit 50 (purifying from gel slices)
- Spin PCR Purification Kit (purifying PCR products)
Microbiology supplies
- LB amp -- Cold Room
- Amp stock -- -20 Fridge
- VWR Petri Dish (100x15mm, case of 500) (for pouring plates)
- Test Tube, Culture (Polystyrene, 12x75, case of 1000) (special tubes for FACS)
Bio Bricks
- R0053 -- Amp -- KBlab -- 4°C
- R0040 -- Amp -- KBlab -- 4°C
- R0011 -- Amp -- KBlab -- 4°C
- Q04121 -- Kan -- KBlab -- 4°C
- Q04510 -- Kan -- KBlab -- 4°C
- Q04400 -- Kan -- KBlab -- 4°C
- E0420 -- Amp -- KBlab -- 4°C
- E0430 -- Amp -- KBlab -- 4°C
- Z0251 -- still in planning stage
- I13504 -- still locating
Planning Bio Bricks
- BBa_J07005 [msr] E.coli JM83 alkaline phosphatase gene (PhoA) 1416 bp
- BBa_J07006 [tmp] malE 1191 bp
- BBa_J07007 [reg] ctx promoter 145 bp
- BBa_J07008 [tmp] ToxR w.t. (from Vibrio Cholarae, aa's 1-294) 1160 bp
- BBa_J07009 [reg] ToxR' (aa's 1-210) 630 bp
- BBa_J07010 [reg] ToxR inner (aa's 1-198; cytoplasm + TM) 594 bp
- BBa_J07011 [rpt] ctx promoter :: gfp reporter 871 bp
- BBa_J07012 [sig] scFv anti-fluorescein [4-4-20][1/3] 762 bp
- BBa_J07013 [sig] scFv anti-fluorescein [4M5.3][2/3]
- BBa_J07014 [sig] scFv anti-fluorescein [S101A][3/3]
Primers
- scFv (pCT-4-4-20, pCT302 S101A, pCT-4M5.3 anti-fluorescein plasmids)
FWD: 5' CATCAT GAATTC GCGGCCGC T TCTAGA GACGTCGTTATGACTCAAACACC 3' T_m,i=71.8 T_m,f=88.5 rand EcoRI NotI XbaI Primer
REV: 5' GCTCAT ACTAGT TTATTA CTTATCATCATCATCCTTATAATC GGAGACGGTGACTGAGGTTCC 3' T_m,i=74.7 T_m,f=87.1 rand SpeI Stop FLAG Tag Primer
- FecA
fecA-Fwd 5’CATCAT GAATTC GCGGCCGC TCTAGA TGACGCCGTTACGCGTTTTTCG 3’ T_m,i=73.2 T_m,f=89.6 rand EcoRI NotI XbaI primer fecA-Rev 5’GCCATA ACTAGT TTATTA TCAGAACTTCAACGACCCCT 3’ T_m,i=68.2 T_m,f=79.9 rand SpeI stop primer
Middle Fwd- 5'GGACGGCAGCGGACTGCAAGTAAAACCGCTGGGAAATAAC 3' T_m=84.7 Middle Rev- 5’GTTATTTCCCAGCGGTTTTACTTGCAGTCCGCTGCCGTCC 3' T_m=84.7
fecA Promoter Fwd- 5’CATCAT GAATTC GCGGCCGC T TCTAGA TGGATAAACATTTCACCACTG 3' T_m,i=65.0 T_m,f=86.2 rand EcoRI NotI XbaI primer fecA Promoter Rev- 5'GCCATA CTGCAG CGGCCGC T ACTAGT CACAAGTCGATACTCAGCTTG 3' T_m,i=68.9 T_m,f=89.8 rand PstI NotI SpeI primer
- FecR
fecR-Fwd 5' CATCAT GAATTC GCGGCCGC TCTAGA tgaatcctttgttaaccgattccc 3'T_m 66 rand EcoRI NotI XbaI primer fecR-Rev 5' GCCATA ACTAGT TTATTA ttacagtggtgaaatgtttatccag 3' T_m 68 rand SpeI stop primer Middle Fwd 5'ctggtgaaagcctacagttcagcgcctcag3' T_m 94 Middle Rev 5'ctgaggcgctgaactgtaggctttcaccag3' T_m 94
- FecI
fecI-Fwd 5'CATCAT GAATTC GCGGCCGC T TCTAGA tgtctgaccgcgccactacc3' T_m 68 rand EcoRI NotI XbaI primer fecI-Rev 5'GCCATA CTGCAG CGGCCGC T ACTAGT A TTATTA tcataacccatactccagacgg3' T_m 66 rand PstI NotI SpeI stop primer
- MalE (updated/fixed)
fwd: 5' CCGTTA GAATTC GCGGCCGC T TCTAGA atgaaaataaaaacaggtgcacgc3' T_m,i=68.8 T_m,f=85.5 junk EcoRI NotI XbaI Primer * rev: 5' ACTGAG CTGCAG GCGGCCGC T ACTAGT TTATTA TTACTTGGTGATACGAGTCTGC 3' T_m,i=69.5 T_m,f=89.4 junk PstI NotI SpeI Stop Primer
- PhoA
fwd: 5' CATGAG GAATTC GCGGCCGC T TCTAGA ATGAAACAGAGCACCATTGCG 3' T_m,i=68.9 T_m,f=88.8 junk EcoRI NotI XbaI Primer rev: 5' AAGCTA CTGCAG GCGGCCGC T ACTAGT TTATTA TTATTTCAGGCCCAGCGCC 3' T_m,i=69.7 T_m,f=89.8 junk PstI NotI SpeI Stop Primer
Middle(1) fwd: 5' caccgcggaactgcaagatgcgaccccggcg 3' T_m=88.8 * rev: 5' CGCCGGGGTCGCATCTTGCAGTTCCGCGGTG 3' T_m=88.8 *
Middle(2) fwd: 5' aggcttttttctgcaagtggaaggcgcgagc 3' T_m=82.2 * rev: 5' GCTCGCGCCTTCCACTTGCAGAAAAAAGCCT 3' T_m=82.2 *