SBB11Ntbk-JessicaWen

Jessica Wen 13:37, 1 March 2011 (EST)

Ligation

Procedure
1.Set up the following reaction:

*6.5uL ddH2O

*1uL T4 DNA Ligase Buffer (small red or black-striped tubes)

*1uL Vector digest

*1uL Insert digest

*0.5uL T4 DNA Ligase

2. Pound upside down on the bench to mix
3. Give it a quick spin to send it back to the bottom of the tube
4. incubate on the benchtop for 30min
5. Put on ice and proceed to the transformation

Transformation

Procedure
1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water to the cells (if greater volume is desired)
3. Add 30 uL of KCM to the cells
4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
5. Add 70 uL of the cell cocktail to the ligation, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42 (longer incubation may work better)
8. Put back on ice for 1 min
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

Jessica Wen 13:53, 24 February 2011 (EST)

Zymo Cleanup of Digest

Procedure
1. transfer into the Zymo column inside a collection tube (small clear guys)
2. spin through, discard waste.
3. Add 200 uL of A4 Wash Bbuffer (which is basically 70% ethanol)
4. spin through, discard waste.
5. Add 200 uL of A4 Wash Buffer
6. spin through, discard waste.
7. spin for 90 seconds, full speed to dry.
8. elute with water into a fresh Eppendorf tube

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 at 55 degrees C 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