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Team:UCL/Science/Proto
From 2014.igem.org
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<b>Notes</b><br/> | <b>Notes</b><br/> | ||
| - | + | <li> The volume of DNA to be digested will depend on the concentration obtained from miniprep of the DNA from culture. This must be made up to 500ng for each 50ul digestion<br/> | |
eg. if concentration BBa_R0010 is available at a concentration of 330ng/ul<br/> | eg. if concentration BBa_R0010 is available at a concentration of 330ng/ul<br/> | ||
we require 500/330 = 1.5ul of the DNA solution for a digestion<br/> | we require 500/330 = 1.5ul of the DNA solution for a digestion<br/> | ||
| - | + | <li> All digestions carried out for iGEM purposes i.e. BioBrick creation are double digests. NEBuffer 2.1 is the ideal buffer for all combinations used.<br/> | |
| - | + | <li> Depending on whether the part being digested is an upstream insert, downstream insert or plasmid backbone, the combinations of enzymes differ. <br/> | |
For Upstream parts, such as promoters digest with EcoRI-HF and SpeI <br/> | For Upstream parts, such as promoters digest with EcoRI-HF and SpeI <br/> | ||
Digest downstream parts with XbaI and PstI<br/> | Digest downstream parts with XbaI and PstI<br/> | ||
Digest plasmid backbones with EcoRI-HF and PstI<br/> | Digest plasmid backbones with EcoRI-HF and PstI<br/> | ||
| + | <br> | ||
<b>Procedure</b><br/> | <b>Procedure</b><br/> | ||
Revision as of 18:04, 17 October 2014
- Creating competent cells
- Digestion
- Ligation
- Making LB Agar Plates
- Transformation
- Agarose Gel Electrophoresis
- Colony Boil
Creating competent cells
LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)
Procedure
1. Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC
2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.
Shake @ 37oC for 1.5-3 hours.
Or
1. Inoculate a single colony into 25ml LB in a 250ml bottle in the morning
2. Shake @ 37oC for 4-6 hours.
Then…
3. Put the cells on ice for 10mins (keep cold from now on).
4. Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.
5. Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).
6. Incubate on ice x 20 minutes
7. Centrifuge as in 2.
8. Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.
9. Dispense in microtubes (300ųl/tube). Freeze at -80oC.
Note: Home-made competent cells were used to transform registry BioBricks
Digestion
MilliQ water, NEBuffer 2.1, BSA, Pipettes and autoclaved tips Enzymes: EcoRI-HF, SpeI, XbaI and PstI,
Notes
eg. if concentration BBa_R0010 is available at a concentration of 330ng/ul
we require 500/330 = 1.5ul of the DNA solution for a digestion
For Upstream parts, such as promoters digest with EcoRI-HF and SpeI
Digest downstream parts with XbaI and PstI
Digest plasmid backbones with EcoRI-HF and PstI
Procedure
1. Calculate amount of DNA solution that will be needed for 500ng
2. Following this, calculate the amount of water needed to make up to 50ul if both enzymes and BSA are 1ul each and Buffer is 5ul.
3. Add the components to a microcentrifuge tube in the order: Water > Buffer > BSA > DNA > Enzymes
4. Leave to incubate at 37ºC for 1-2 hours
5. Remove and deactivate the enzymes by incubating at 80ºC for 20 minutes
Ligation
Making LB Agar Plates
LB Agar powder, plates, antibiotics, sterile jar, sterile flame or biosafety cabinet.
Procedure
1. Dissolve LB agar in sterile/autoclaved jar of sterile water 40g/L.
2. Swirl until completely dissolved
3. Autoclave LB agar
The following steps should be completed under a sterile flame or biosafety cabinet. Ideally after autoclaving, before LB Agar solidifies:
4. Add antibiotics from their stock solutions in 1:1000 ratio with LB agar, can be added either directly to each individual plate or to the entire solution.
5. Pour agar into plates, roughly half way (30-50mL per plate). Leave plates with lids half on (prevent condensation).
6. Allow plates to solidify under flame
7. Cover and leave on bench or in fridge
Plasmid DNA Transformation
Competent Cells, Plasmid DNA, Antibiotic Plates
Procedure
1. Thaw competent cells on ice
2. 50uL cells enough for 1 transformation
3. Add 1ug of DNA to 50uL competent cells
If biobrick from distribution, resuspend DNA well in 10uL ddH20
4. Add 2-5uL DNA i.e. ligation product or BioBrick re-suspension to 50uL competent cells
5. Flick by hand or pipette up and down gently
6. Place cells on ice for 30 minutes
7. Place cells in water bath or heat block at 42oC for 30 seconds
8. Place cells on ice for 2 minutes
9. Add 950ul of SOC or LB media and place in a shaking incubator for a maximum of 2 hours (37oC/250rpm) 10. Label two petri dishes with LB agar and the appropriate antibiotics(s) with the date, plasmid backbone and ligated genes/parts and antibiotic added.
You may also choose to write down the volume of transformed product plated
11. Spin down the incubated cells for 2mins at 4000rpm to obtain a cell pellet.
12. Pipette 100ul of the supernatant or fresh LB media before discarding the rest of the supernatant and re-suspending the pellet in the 100ul of media
13. Incubate the plates at 37ºC for 12-14 hours, always place plates lid-down.
If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria
You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.
