User:Jessica Karen Wong/Construction

Output Measurement Kit

1. PCR T9002 with MfeI site on FWD primer tail and NsiI on REV primer tail.
   • Design a forward and reverse primer to match T9002 with tails Mfe1 for the forward and Nsi1 for the reverse
2. Digest backbone 3K3 with Eco and Pst
3. Digest T9002 with Mfe and Nsi
4. Do a 2 way ligation between T9002 and the backbone
5. Do a transformation to find the successfully ligated product
   • Colony PCR and sequencing
   • This scars the BB sites from the vector.
6. PCR vector with EcoR1 site on FWD primer tail and PstI on REV primer tail.
   • Design a forward and reverse primer to match T9002 with tails Pst for the forward and EcoR1 for the reverse
   • Cut the scarred T9002 with EcoR1 and Pst
   • Miniprep the CCDB and digest with EcoR1 and Pst
   • Ligate the CCDB (P1010) and the scarred T9002
   • Transform into DB3.1
   • This is the same method the Registry uses to make backbone construction vector.
7. 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 File:I2055 synthesis final.doc

• 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