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:
- 1µl Ligase Buffer (NEB)
- 1µl T4 Ligase
- 1µl Vector (pUA66-X-B-2)
- 7µl Insert (kateE-X-B-2)
- Leave at room temperature for 2 hours
Prepare 190 bottles of 10 ml LB broth
I prepared 190 bottles of 10ml LB broth
- 12g LB-broth powder
- Add 400ml of water
- Distribute in 190 bottles - about 10ml discrepancy
- Autoclave at 121ºC, 15psi for 15 minutes
Prepare 6 bottles of 200 ml LB Agar
For each bottle I added
- 6.4g LB-agar powder
- Add 200ml of water
- Autoclave at 121ºC, 15psi for 15 minutes
Prepare 190 bottles of 10 ml LB broth
I prepared 190 bottles of 10ml LB broth
- 12g LB-broth powder
- Add 400ml of water
- Distribute in 190 bottles - about 10ml discrepancy
- Autoclave at 121ºC, 15psi for 15 minutes
Prepare 6 bottles of 200 ml LB Agar
For each bottle I added
- 6.4g LB-agar powder
- Add 200ml of water
- Autoclave at 121ºC, 15psi for 15 minutes
Digestion pUA66katE#A1 BamHI
- 15µl of pUA66katE#A1
- 0.3µl 100xBSA
- 3µl 10xNEB#3
- 11.7µl water
- 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%)
- Weigh out 0.6g Agarose powder
- Add 60ml 1xTAE
- Heat in microwave until no more specs and cool
- Add 2µl Ethidium Bromide
- Pour in prepared tray and wait until ready
Loading 1%
- Lane 1: 10µl, 100bp NEB Quick Ladder
- Lane 2: 30µl, pUA66katE#A1 BamHI
- Lane 3-8: BLANK
- Run at 100V, 400mA, 60 minutes
Gel Picture
Transformation, Ligation pUA66katE
Before:
Place SOC in 37ºC to prewarm
- Take 50µl of competent cell and leave on ice for 5 minutes
- Add all of the ligase reaction (10µl)
- Leave on ice for 5 minutes
- Place in a 42ºC water bath for 1 minute
- Place back on ice for 5 minutes
- Add 250µl SOC
- Incubate in shaker for 1 hour at 37ºC
- Add to fresh Kanamycin plate and spread with glassbeads
- Place in incubator overnight
Transformation, pBestLuc
Before:
Place SOC in 37ºC to prewarm
- Take 50µl of competent cell and leave on ice for 5 minutes
- Add 1µl pBestLuc
- Leave on ice for 5 minutes
- Place in a 42ºC water bath for 1 minute
- Place back on ice for 5 minutes
- Add 250µl SOC
- Incubate in shaker for 1 hour at 37ºC
- Add to fresh Ampicillin plate and spread with glassbeads
- 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:
- Add 0.3µl of 100xBSA
- Add 3µl of 10xNEB#Ω3
- Add 15µl of pUA66katE#A1
- Add 11.7µl Water
- 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:
- 1µl Ligase Buffer (NEB)
- 1µl T4 Ligase
- 1µl Vector (pUA66-X-B-2)
- 7µl Insert (kateE-X-B-2)
- Leave at 37ºC overnight
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