User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/16

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Ligation pUA66 XhoI/BamI with katE XhoI/BamHI

Setting up a ligation based on pUA66-X-B-2 (Lane 6) User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14#Running Gel of purified products and katE-X-B-2 (Lane 2) User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14#Running Gel of purified products

Reaction:

  1. 1µl Ligase Buffer (NEB)
  2. 1µl T4 Ligase
  3. 1µl Vector (pUA66-X-B-2)
  4. 7µl Insert (kateE-X-B-2)
  5. Leave at room temperature for 2 hours

Prepare 190 bottles of 10 ml LB broth

I prepared 190 bottles of 10ml LB broth

  1. 12g LB-broth powder
  2. Add 400ml of water
  3. Distribute in 190 bottles - about 10ml discrepancy
  4. Autoclave at 121ºC, 15psi for 15 minutes

Prepare 6 bottles of 200 ml LB Agar

For each bottle I added

  1. 6.4g LB-agar powder
  2. Add 200ml of water
  3. Autoclave at 121ºC, 15psi for 15 minutes

Prepare 190 bottles of 10 ml LB broth

I prepared 190 bottles of 10ml LB broth

  1. 12g LB-broth powder
  2. Add 400ml of water
  3. Distribute in 190 bottles - about 10ml discrepancy
  4. Autoclave at 121ºC, 15psi for 15 minutes

Prepare 6 bottles of 200 ml LB Agar

For each bottle I added

  1. 6.4g LB-agar powder
  2. Add 200ml of water
  3. Autoclave at 121ºC, 15psi for 15 minutes

Digestion pUA66katE#A1 BamHI

  1. 15µl of pUA66katE#A1
  2. 0.3µl 100xBSA
  3. 3µl 10xNEB#3
  4. 11.7µl water
  5. Place in incubator for 2 hours at 37ºC

Gel, size of pUA66katE#A1 single digest

I prepared a 1% gel to check if the the plasmid was a mega vector - it wasn't.

Gel (1%)

  1. Weigh out 0.6g Agarose powder
  2. Add 60ml 1xTAE
  3. Heat in microwave until no more specs and cool
  4. Add 2µl Ethidium Bromide
  5. Pour in prepared tray and wait until ready

Loading 1%

  1. Lane 1: 10µl, 100bp NEB Quick Ladder
  2. Lane 2: 30µl, pUA66katE#A1 BamHI
  3. Lane 3-8: BLANK
  4. Run at 100V, 400mA, 60 minutes

Gel Picture 16082010-pUAkatEA1-BamHI.jpg

Transformation, Ligation pUA66katE

Before:

Place SOC in 37ºC to prewarm

  1. Take 50µl of competent cell and leave on ice for 5 minutes
  2. Add all of the ligase reaction (10µl)
  3. Leave on ice for 5 minutes
  4. Place in a 42ºC water bath for 1 minute
  5. Place back on ice for 5 minutes
  6. Add 250µl SOC
  7. Incubate in shaker for 1 hour at 37ºC
  8. Add to fresh Kanamycin plate and spread with glassbeads
  9. Place in incubator overnight

Transformation, pBestLuc

Before:

Place SOC in 37ºC to prewarm

  1. Take 50µl of competent cell and leave on ice for 5 minutes
  2. Add 1µl pBestLuc
  3. Leave on ice for 5 minutes
  4. Place in a 42ºC water bath for 1 minute
  5. Place back on ice for 5 minutes
  6. Add 250µl SOC
  7. Incubate in shaker for 1 hour at 37ºC
  8. Add to fresh Ampicillin plate and spread with glassbeads
  9. Place in incubator overnight

Single digestion pUA66katE#A1

I am setting up this single digestion to verify that the ligation has produced a mega vector as opposed to it being plasmid contamination.

For a final volume of 30µl:

  1. Add 0.3µl of 100xBSA
  2. Add 3µl of 10xNEB#Ω3
  3. Add 15µl of pUA66katE#A1
  4. Add 11.7µl Water
  5. Leave in 37ºC for 2 hours


Ligation #2 pUA66 XhoI/BamI with katE XhoI/BamHI - overnight

Setting up a ligation based on pUA66-X-B-2 (Lane 6) User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14#Running Gel of purified products and katE-X-B-2 (Lane 2) User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14#Running Gel of purified products

Reaction:

  1. 1µl Ligase Buffer (NEB)
  2. 1µl T4 Ligase
  3. 1µl Vector (pUA66-X-B-2)
  4. 7µl Insert (kateE-X-B-2)
  5. Leave at 37ºC overnight