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+ | <li> Add 2ul of AP Buffer </li> | ||
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+ | <td> Content </td> | ||
+ | <td> ul </td> | ||
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+ | <td> AP Buffer </td> | ||
+ | <td> 2 </td> | ||
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+ | <td> Phosphatase </td> | ||
+ | <td> 1 ul</td> | ||
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+ | <td> Plasmid </td> | ||
+ | <td> 10 </td> | ||
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+ | <td> H<sub>2</sub>O</td> | ||
+ | <td> 7 </td> | ||
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Latest revision as of 03:06, 12 October 2016
Protocols & Experiments
Agarose Gel Preparation
- Add 50mL of 1X TAE buffer to a 500mL erlenmeyer flask
- Add 0.5g of 1% agarose
- Microwave for three 20 second intervals and cool slightly
- Add 1ul Ethidium Bromide
- Mix and pour into gel box. Add Comb
- Cool for 30 minutes
YPD Media
-
For liquid media
- Add 20g of Bacto peptone, 10g of yeast extract and 950mL of water to a flask
- Autoclave on the liquid setting
- Add 50mL of 40% glucose and mix
- Add same reagents as liquid, plus 24g of Bacto agar
- Autoclave
- Stir on a magnetic stir plate and add 50ml of 40% glucose
- Pour into petri dishes
For solid media
LB Broth
Add items to flask:
- 5g Tryptone
- 2.5g Yeast Extract
- 5g NaCl
- H2O filled to 500mL
- Mix with magnetic stir bar on low heat, autoclave for liquids
Nanodrop
- Select Nucleic Acid
- Clean, drop 1ul water, press okay
- Blank with 1ul
- Measure with 1ul of substance
- 260/280 ratio should be near 2
LB Agar
Add items to flask:
- 5g Tryptone
- 2.5g Yeast Extract
- 5g NaCl
- 7.5g Agar
- H2O filled to 500mL
- Mix with magnetic stir bar on low heat, autoclave for liquids
- Monarch Plasmid Miniprep Kit
- Monarch DNA Gel Extraction Kit
- Monarch PCR & DNA Cleanup Kit
NEB Monarch Nucleic Acid Purification Protocols
The following NEB kits were used and protocols can be found on their website.
Digest
- Add Nuclease free water to a 2ml test tube
- Add 10X buffer for restriction enzymes
- Add restriction Enzymes
- Add DNA using (Need 500ng of DNA for 25ul)
- Make negative controls without the enzymes
- Make up volume with nuclease free water
- Amounts of reagents listed below
50ul Reaction
Content | ul |
---|---|
Nuclease-free water | Bring to volume |
10X Buffer | 5 |
Restriction Enzymes | 1 each |
DNA | Calculate volume for DNA |
10ul Reaction
Content | ul |
---|---|
Nuclease-free water | Bring to volume |
10X Buffer | 5 |
Restriction Enzymes | .5 each |
DNA | Calculate volume for DNA |
Transformation
- Thaw competent E. coli cells on ice and pipette 50ul cells into a transformation tube
- Add 1-5ul containing 1pg-100ng of plasmid DNA and flick tube 4-5 times
- Place tube on ice for 30 minutes
- Heat shock at 42°C for 30 seconds
- Place on ice for 5 minutes
- Add 950ul room temperature SOC to mixture
- Incubate at 37°C for 60 minutes at 250rpm
- Mix and perform several 10-fold serial dilutions in SOC
- Spread 50-100ul onto a plate and incubate overnight at 37°C
Ligation
Content | ul |
---|---|
Nuclease-free water | Bring to volume |
10X T4 Ligase Buffer | 2 |
T4 Ligase | 1 |
Vector : Insert Ratio | 1:3 |
- There must be 3x as much insert as vector
- Total volume of solution must be 20ul
- Negative control contains no inserts
Phosphatase
- Add 1pmol of DNA ends
- Add 2ul of AP Buffer
Content | ul |
AP Buffer | 2 |
Phosphatase | 1 ul |
Plasmid | 10 |
H2O | 7 |