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 and katE-X-B-2 (Lane 2) User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14

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

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 and katE-X-B-2 (Lane 2) User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14

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


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