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pSB1C3-LovTAP and pCEP4-HA-RO colony PCR
Protocol: PCR
PCR is a reaction that makes it possible (and relatively easy) to amplify
a certain region of DNA. The first step is the selection of that region
(and the design of the relevant primers). Primer design can be done by hand, or by
using our Primer Design Helper. Once
done, order the primers (in our case, we ordered from them [http://www.idtdna.com/ IDT]).
When you've received the primers, prepare them and make sure you've got your PCR kit (we used the "Phusion® High-Fidelity DNA Polymerase"). Start preparing your master mix, the composition for one tube is:
1X Mastermix 20μl reaction, add in this order
| Reagent | Volume [μl] |
|---|---|
| Water | Complete to total volume of 20μl |
| HF-Buffer (5x) | 4 |
| DMSO (optional) | 0.6 |
| dNTPs | 0.4 |
| Forward primer (50μM) | 0.2 |
| Reverse primer (50μM) | 0.2 |
| Template (10ng/μl) | 0.5 |
| Phusion HF polymerase | 0.2 |
Prepare one or two extra tubes-worth of reagent (you'll use some liquid on the walls of your tips).
Once you've finished, you should run the resulting products on a gel to check if everything went as planned.
Tips
- Thaw the HF-Buffer, DMSO and dNTPs before making the mastermix.
- Avoid taking the Phusion-HF polymerase out of the freezer (only take it out briefly when you need to add it).
- If the reactions have different primers and/or template, add the polymerase right after the dNTPs, split the mastermix and add the rest.
- Don't forget positive and negative controls
- Primers should have similar Tms (less than 5°C).
- Primer Tm calculation is a less exact science than it should be (just test several tools and compare their results). If you're not sure what the correct Tm is, consider using a gradient PCR.
- Avoid primers with strong secondary structures.
- PCR can introduce mutations. Don't forget to sequence your final product (this could be your final plasmid): you really don't want to lose a few weeks because of a "corrupt" plasmid.
A colony PCR was performed on the (hopefully) correctly ligated LovTAP BioBrick from the day before, and on the pCEP4-HA-RO plates that were stored in the fridge.
Gel electrophoresis
Protocol: Gel Electrophoresis
Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%.
VOL is the desired volume of gel in ml:
CH Lab
- Add 0.01*VOL g of agarose to a clean glass bottle.
- Pour VOL/50 ml of 50xTAE in a graduated cylinder. Fill up to VOL ml with di water.
- Add the resulting VOL ml of 1xTAE to the glass bottle with agarose.
- Microwave, at 7, the bottle (loose cap!) until it boils.
- Carefully remove bottle (can be super heated!) and check for the total absence of particles. Microwave again if needed.
- Prepare a gel box, with comb, and fill it up with the agarose solution (maybe not the whole solution is needed).
- Add 0.05 µl per ml of gel in the box of Red Gel (it's in the iGEM drawer) and stirr until disolved.
- Wait until cold and solidified.
- Carefully remove comb.
- Place the box in the electrophoresis chamber.
- Fill up the electrophresis chamber with 1x TAE buffer.
- Add blue dye to the DNA samples (6x loading buffer, that is 10 µl in 50 µl of DNA solution).
- Inject 30 µl of ladder marker in the first well (that's 1 µg of DNA).
- Inject 60 µl of each DNA solution in the other wells.
- Set voltage to 70-90 V and run for 30-40 min, or until the dye reaches the last 25% of the gel length (DNA travels from - to +).
- Place the gel under the camera, cover, turn UV on and take photos!
Preparing the ladder:
- get 1kb ladder DNA from the freezer (500 µg/ml).
- for 30 charges, 30 µl per charge, we need 900 µl:
- 60 µl of 1kb ladder DNA
- 150 µl of dye (6x loading buffer)
- 690 µl of water
BM Lab
In this lab the gels are slightly different. The total volumes for the small, the medium and the large gel are respectively 60ml, 80ml and 90ml. As we use 0.5x TAE buffer instead of 1x, we can use higher voltages (170V seems to work fine). The gel should run 20-40 minutes, not more. As the gel is thinner, load less DNA (up to ~10ul).
- pSB1C3-LovTAP gel
This is a working BioBrick, all the ligations have worked!
- pCEP4-HA-RO gel
The colony PCR has failed. It turned out that the primers we had designed for this check were wrong. We ordered new ones and recultivated the colonies during the wait.
Maxipreps
Protocol: MaxiPrep
The evening before, take a big Erlenmeyer (at least 1L) and put 200ml LB in it. Add the appropriate antibiotics at the correct concentration (ampicilin: 200ul of 100mg/ml solution). Put in bacteria from a single colony of a freshly streaked plate or from a glycerol stock (warning: taking bacteria from glycerol stock seems to cause them to start growing later - due to thawing? - add one-two hours to the incubation time). Put them in the incubator for 14-15 hours (the contents of the bottle should be yellow-ish between translucid and opaque).
We then use the MaxiPrep kit (Plasmid DNA Purification kit) and protocol from Macherey-Nagel.
The complete handbook can be found [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NuBo.pdf here]. We usually use the protocol that starts at page 24 for "Maxi".
Maxipreps were made from overnight cultures of pHY42, pcDNA3.1(+)-LovTAP, and pGL4.30-GFP.
Western Blot result from 4.sep.12
Protocol: Western Blot
Gel Ingredients (choose percentage according to the size of the protein)
| 4-40 kDA | 20% |
| 12-45 kDA | 15% |
| 10-70 kDA | 12.5% |
| 15-100 kDA | 10% |
| 25-200 kDA | 8% |
| Separating gel | |
| Gel percentage | 7.5 % |
| 30% Polyacrylamide | 10 mL |
| 1.5M Tris (pH 8.8) | 10 mL |
| 10% Ammonium persulfate | 0.4 mL |
| 10% SDS | 0.4 mL |
| TEMED | 0.038 mL |
| H2O | 19.2 mL |
| Total volume | 40 mL |
| Stacking gel | |
| Gel percentage | 5 % |
| 30% Polyacrylamide | 1.36 mL |
| 1M Tris (pH 6.8) | 1 mL |
| 10% Ammonium persulfate | 0.08 mL |
| 10% SDS | 0.08 mL |
| TEMED | 0.008 mL |
| H2O | 5.44 mL |
| Total volume | 8 mL |
Preparing Protein Samples
1. Centrifuge around 5 million cells (of any volume) at 2,500 rpm for 10 min.
2. Discard the supernatant with a vacuum pump.
3. Resuspend the cell pellet with 1x PBS and centrifuge it at 2,500 rpm for 10 min.
4. Discard the supernatant with a vacuum pump.
5. Add appropriate amount of lysis buffer depending on the pellet size (for a 20 mg pellet, 150 µl of IP lysis buffer).
6. Keep the lysed sample on ice for 10 min - flick every 3 minutes.
7. Add 3x SDS lysis buffer (for a 20 mg pellet, 75 µl).
8. Incubate the sample for 5 minutes at 95 degrees, to denature proteins.
Preparing loading samples
1. Load the ladder (7 µl is the recommended volume).
2. Complete sample volume to 50 µl.
3. Load the samples.
I. SDS Gel electrophoresis
1. Prepare the separating and stacking gel solutions without APS and TEMED.
2. Add APS and TEMED to the separating gel solution only when the SDS kit is ready to be used, they are time-sensitive. Move the solution inside of the setup. Add some distilled water on top of it.
3. After 20-30 mins, remove the water and check whether the gel has solidified. Don't move to the next step until it does.
4. Add TEMED to the stacking gel solution, pour it on top of the solidified separating gel.
5. Insert a stack carefully and leave it for 20-30 mins.
6. Take the stack out and fill the kit with SDS loading buffer.
7. Load the samples.
8. Add more loading buffer, set the voltage to 80 Volts. Leave for 1.5 hours.
II. Membrane transfer
1. Prepare a membrane transfer kit.
2. Take the gel out of the SDS kit and put it on the membrane paper.
3. From bottom to top, assemble the components in the following order: 1) Sponge - 2) Blot paper - 3) Membrane - 4) Gel (Pour some M-transfer buffer on the gel) - 5) Blot paper again - 6) Sponge again.
4. Close the sandwich, set the voltage to 20 V. Leave for 30 mins - 1 hour.
5. Discard the gel. Leave the membrane in 5% skim milk with 30ml of TBST buffer (blocking buffer, to achieve the 5%, add 1.5 g of skim milk powder to the buffer) for one hour.
III. Antibody tagging
1. Discard the blocking buffer, leave only 5ml of it. Add primary antibody with a ratio of 1:1000 or 1:2000 (5 µl of antibody in 5 ml of buffer gives 1:1000)
2. Leave the mix overnight at 4 °C.
3. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).
4. Dilute the secondary antibody (for example, goat anti-rabbit antibody) to 1:2000 in 5% skim milk buffer. Add it. Leave at room temperature for 2 hours.
5. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).
6. Reveal the protein bands in the dark room.
VP16 with CHO cell lysate, to check how it looks in cells.
- Lane 1: Ladder
- Lane 2: CHO cell lysate with VP16 protein 5microG
- Lane 3: CHO cell lysate with VP16 protein 2microG
- Lane 4: CHO cell lysate with VP16 protein 1microG
- Lane 5: CHO cell lysate with VP16 protein 500nanoG
- Lane 6: CHO cell lysate with VP16 protein 100nanoG
Looks like the size of the VP16 protein we are testing our WB is around 35kDa. This does not correspond to the 25kDa that the manual promises, we have no idea why but all the time VP16 has shown this size of band in the WB.
Overnight cultures
Protocol: Prepare Plasmid Extraction (culture for Miniprep)
- Select and number colonies on the plates.
- Prepare tubes of LB medium with the correct quantity of antibiotics (100 µg/ml for Amp, Spc or chloramphenicol).
- Amp can be found in the -20ºC freezer at Ecoli, labeled as "stock". It is 100 µg/µl, or 1000x.
- The tubes to be used are the 14 ml round bottom found in front of the iGEM drawers (Falcon). Culture with cap in the first step (loose) and close to the second step after culture.
- Touch each colony with a clean pipette tip and put it in a tube.
- Put in incubator.
Overnight cultures of the pSB1C3-RFP plasmid for miniprepping. It was cultured on chloramphenicol.
Soldering
The assembly of the reactor plates was completed without major issues.
