User:Jessica Karen Wong/Construction

Output Measurement Kit

 * 1) PCR T9002 with MfeI site on FWD primer tail and NsiI on REV primer tail.
 * 2) *Design a forward and reverse primer to match T9002 with tails Mfe1 for the forward and Nsi1 for the reverse
 * 3) Digest backbone 3K3 with Eco and Pst
 * 4) Digest T9002 with Mfe and Nsi
 * 5) Do a 2 way ligation between T9002 and the backbone
 * 6) Do a transformation to find the successfully ligated product
 * 7) *Colony PCR and sequencing
 * 8) *This scars the BB sites from the vector.
 * 9) PCR vector with EcoR1 site on FWD primer tail and PstI on REV primer tail.
 * 10) *Design a forward and reverse primer to match T9002 with tails Pst for the forward and EcoR1 for the reverse
 * 11) *Cut the scarred T9002 with EcoR1 and Pst
 * 12) *Miniprep the CCDB and digest with EcoR1 and Pst
 * 13) *Ligate the CCDB (P1010) and the scarred T9002
 * 14) *Transform into DB3.1
 * 15) *This is the same method the Registry uses to make backbone construction vector.
 * 16) Pre-cut vector could be given to teams, along with a standard characterization procedure

Making a Biobrick out of "A" and "B"

 * Start with cells containing each A and B on an Amp backbone (or overnight from a glycerol)
 * Miniprep A and B
 * Digest:
 * A with EcoR1 and Spe1
 * B with Xba1 and Pst1
 * The backbone you want in your kit with EcoR1 and Pst1
 * PCR clean-up each digest
 * 3-Way Ligate A, B, and the backbone plasmid
 * Transform and plate on media with same resistance as backbone

Note: Make sure the backbone you choose has a resistance besides the ones that A and B originally had

New T9002 Construction Method

 * 1) PCR F2620 with Mfe FWD tail and Ecor1, Not1, Xba1 (BB prefix) REV tail
 * 2) Cut F2620 Mfe/Xba and cut ccdb backbone Eco/Xba
 * 3) Ligate and transform
 * 4) PCR E0240 with BB suffix (Spe, Not, Pst) FWD tail and Nsi REV tail
 * 5) Cut E0240 Spe/Nsi
 * 6) Cut previous ligation product Spe/Pst
 * 7) Ligate and Transform

Post-Synthesis I2055
Synthesis Proposal
 * Get from company in form: AatII Plasmid__R0040 BsiHKAI___E X____GFP T NsiI
 * Also get Conversion Construct: BsiHKAI__S X___
 * Cut I2055 A/N and Plasmid A/P and ligate - Version 1 (E/X) is finished in both plasmids
 * Cut I2055-3K3 version 1 B/X, cut conversion construct B/X, and ligate - Version 2 is finished in 3K3
 * Cut I2055-3K3 version 2 A/X, cut I2055-1AK3 version 1 A/X and ligate - Version 2 is finished in 1AK3