SBB11Ntbk-JessicaWen

Jessica Wen 13:16, 22 February 2011 (EST)

Eco/Bam Digest of PCR Products

Procedure
1. For each part place in a new PCR tube:

• 8uL PCR Product
  
• 1uL NEB Buffer
  
• 0.5 uL EcoRI
  
• 0.5 uL BamHI
  

2. Place in thermocycler for 1 hour
3. Run preparative gel

• cut out bands place in epindorph tube
  
• add 600uL ADB buffer and place on heat block for 10 min to dissolve
  

Jessica Wen 13:31, 17 February 2011 (EST)

Analytical Gel of PCR Products

Procedure

Gel Preparation In a 0.5mL tube add:
2uL of PCR product
5uL of DNA Loading Buffer

Run Gel

Sb1112 did not work. Run PCR cloning again, one the same as before and one with 10% DMSO.

for sbb1137 and sbb1123 continue with Zymo Cleanup, which removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

Zymo Cleanup
Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of PE or Zymo Wash buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Jessica Wen 14:05, 15 February 2011 (EST)

Cloning by PCR of the following construction files

Part sbb1112 {P_cspI}
PCR jwsbb1112F/ss37f on MG1655 (506bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sb1112 {P_cspI}

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jwsbb1112F Forward Construction of P_cspI promoter part ccaaaGAATTCatgAGATCTATGAATCTTTAAGAGATAAAAAGC
ss37f BglBrick basic part cloning of cspI promoter tttggGGATCCgccatctttcggcgtgatgaaacc

Part sbb1137 {P_yciG}
PCR ss64r/ss64f on MG1655 gen. (616bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1137 {P_yciG}

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ss64fReverse Cloning of P_yciG tttggGGATCCgtcggatgccttctcacggtcttcgg
ss64rForward Cloning of P_yciG aaaccGAATTCatgAGATCTagcagcgattgatgcaggagctgcg

Part sbb1123 {P_rfaH}
PCR ss49r/ss49f on MG1655 gen. (421bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1123 {P_rfaH}

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ss49fReverse Cloning of P_rfaH tttggGGATCCgcatcactgactgcagtacgttttccacgcacg
ss49rForward Cloning of P_rfaH aaaccGAATTCatgAGATCTgcgtgatacgttttagctcaccctg

Procedure
If the oligo is not already in 10uM concentration make an oligo dilution of:
9uL water
100uL uM oligo

Make one PCR reaction tube of the following for each part:
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA