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Revision as of 13:32, 4 August 2009
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Week One
Dear Diary
This week we started lab work. We started by growing some Colis, making some amp-plates, making RIP DNA, making competent cells and trying to transform biobricks.
Day One - Preparation.
On the first day we streaked a strain of E. Coli called Top10 onto 5 LA plates (LA = LB medium with Agarose, LB = Luria-Bertani broth [http://en.wikipedia.org/wiki/Lysogeny_broth]). The plates were incubated at 37 degrees C overnight. [http://en.wikipedia.org/wiki/Streaking_(microbiology) Streaking] was done as shown on the picture [PICTURE FROM NOTEBOOK].
We also made LA plates containing ampicillin as our selection antibiotic. We decided to use an ampicillin plate-concentration of 50 μg/mL based on reported ampicillin resistance of our biobrick containing plasmids. We later found out that the plasmids provide sufficient ampicillin resistance for harsher selection with 100 μg/mL[#]. How to make 50 μg/mL plates.
Day Two - DNA creation and purification.
Today we started creating our RIP DNA. We had previously ordered primers with an overlap in order to make RIP DNA via [http://en.wikipedia.org/wiki/Pcr PCR]. In order to achieve a primer-stock concentration of 100 μM we added diH2O(de-ionized, autoclaved water) to our raw primers:
| Name | Added Water | Description |
|---|---|---|
| Igem-1 | 187μL | RIP with export sequence (sRIP) |
| Igem-2 | 200μL | RIP with export sequence (sRIP) |
| Igem-3 | 301μL | RIP |
| Igem-4 | 269μL | RIP |
Since we dont need all the DNA at once, we diluted the primer solutions to 20 μM, by adding 40 μL of diH2O to 10 μL of primer-stock.
We did PCR with our 20 μM solution of primer using this protocol, due to a shortage of Pfx-enzyme, we used Pfu enzyme instead and didn't add MgSO4 (which the Pfu-buffer already contains). We also elongated at 70 degrees C for 2 minutes. We made 4 tubes for both sRIP and RIP for extra safety.
Inspection of yesterdays platestreakings. We observed growth on all 5 LA plates. Tertiary growth on several plates. 1 plate has been streaked too deeply, it's hard to see colonies.
Post-PCR gel preparation. 4 μL of diH2O and 1μL of loading buffer (containing a colouring agent) was added to 5 μL of PCR product. Process was repeated for all 8 tubes. The solution was applied to a 1,5 % (mass/volume, 4,5g agarose in 300mL of diH2O) agarose gel og run for about 15 minutes, or until the bands reach halfway of the gel. Technically the solution is applied to the gel by inserting the tip of a pipette into the appropriate slot and releasing the material so that it stays in the slot, remember to apply a size-appropriate DNA ladder to a slot for size-determination of samples. [Gel-billede 1].
Tube-inoculation. We applied a colony from our LA-streakings to LB liquid medium and grew it overnight at 37 degress C and shaking. These cells are later made competent.
Gel purification of PCR product. Solution for gel-electroforesis was prepared as described earlier except, we now prepared 50 μL, by adding 5 μL of loading buffer to 45 μL of PCR-product. applying the solution to deeper, wider slots, run the gel as before, excise the bands, and weighing them.
| Name: | sRIP-1 | sRIP-2 | sRIP-3 | sRIP-4 | RIP-1 | RIP-2 | RIP-3 | RIP-4 |
| Weight | 390mg | 420mg | 520mg | 560mg | 719mg | 600mg | 450mg | 430mg |
We now followed a purification protocol, complete with kit. To the eppendorf-tubes containing the excised bands we added 1 μL of capture buffer per mg gel, heated to 60 degrees C and vortexed until the bands had been dissolved. From the tubes we transferred 600 μL to microspin tubes (w/ filter) and centrifuged for 30 seconds at 14500 RPM to capture the DNA in the filter. Flowthrough was discarded. From here on, as protocol describes.
Day Three - Competence and transformation.
We started the day by making competent cells according to this protocol. We measured OD550 (optical density at 550nm wavelength) as:
| Time | #1 | #2 |
|---|---|---|
| 09:00 | 0,036 | 0,040 |
| 10:15 | 0,08 | 0,115 |
| 10:45 | 0,167 | 0,253 |
We used 1mL pure LB medium as reference.
The PCR product we purified yesterday was tested in the same way as the product was before purification. [Gel-billede 2].
Using the competent cells from earlier and this protocol we attempted to transform our Colis with biobrick plasmids B0015, B0034, R0011, J23100, pSB1A3 (containing p1010) and 1 negative control (without plasmid). 1 hour incubation time on 37 degrees C.
4/8
Dear diary, today we are beginning to get ahold of the last week and a halfs chaos.
Purification of PCR double-bricks
PCR product RIP-23L and sRIP-23L was mixed with 5uL loading buffer og purified from 1,5%(w/V) agarose gel.
Bands excised and dissolved:
RIP-23L 55mg
sRIP-23L 114mg
Purified PCR product are stored on -20 degress C.
Gel of yesterdays restrictions
| Content | Expected length | Band 1 | Band 2 | Band 3 | Smear (+/-) | |
|---|---|---|---|---|---|---|
| 1 | sRIP | 174 | ~150bp | - | ||
| 2 | 23L | 129 | >2kbp | ~170bp | - | |
| 3 | pSB1C3+J04450 | >3kbp | >2kbp | ~2kbp | ~1,4 kbp | - |
| 4 | RIP | - | ||||
| 5 | 6G | >10kbp | >2kbp | + | ||
| 6 | 2M | ~10kbp | >2kbp | + | ||
| 7 | 13B | >2kbp | - |
1,2 and 3 look promising, the rest, not so much.
Ligations
1+2+3 → 11 = sRIP-23L-pSB1C3
4+2+3 → 12 = RIP-23L-pSB1C3
5+6+3 → 13 = 6G-2M-pSB1C3
7+3 → 14 = 13B-pSB1C3
Nothing really about the ligation process...
Ligation gel
| Wanted length | Band 1 | Band 2 | Band 3 | Smear (+/-) | |
|---|---|---|---|---|---|
| 1 | 2299 | ~3,5kbp | ~2,2kbp | ~1,2kbp | + |
| 2 | 2239 | ~4,0kbp | ~2,2kbp | ~1,2kbp | + |
| 3 | 2147 | ~5kbp (very smeary) | ~2,2kbp | ~1,2 kbp | - |
| 4 | 2146 | ~3,5kbp | 2,2kbp | 1,2kbp | - |
Electroporation of ligationproducts
12 exploded :(
Purification of double-brick RIP-23L
This brick was expected at 313bp, but the closest visible band was at ~250-300bp (the other band was at <50bp). The area from the visible band and up to where the brick was expected was excsed and split equally into to tubes for purification. Stored overnight.
Week 2
Dear Diary
This week we started working on the Wiki and continued our labwork. In the lab we had to start our transformation over, since we didn't have any usable growth in last weeks transformed cells. In the second round of transformation we added a second protocol (electroporation) and made minor changes to the classic protocol.
Day One - Starting over.
Transformation control. Sadly, we observed no growth on most of the plates from our first transformation. Only the plates containing biobrick B0015 (the double terminator in plasmid pSB1AK3) showed clumpy growth [images], 3 out of 4 plates (both 5 μL and one 2 μL) showed growth. One possible explanation attributed these observations to an uneven distribution of ampicillin in the medium, most likely though is that our competent cells werent quite so competent. Nevertheless we needed to start over with the transformation.
Amp preparation. We added 5 mL of diH2O to NEW powder ampicillin and vortexed to dissolve, creating an amp concentration of 100mg/mL. We transferred 1mL to each of three eppendorf-tubes. From the tubes we transferred 0,25 mL to ~500mL of LA medium, creating a ~1:2000 concentration gradient, with a medium concentration of amp = 50 μg/mL, to evenly distribute the amp in the medium it was shaken and rotated vigourosly.
Due to a mysterious lack of liquid in well 23L(B0015, double terminator) we decided to dilute all open wells (2M, 3K, 6G, 18C, 23L) with 2 μL of diH2O.
Electroporation. We made 7 samples, 5 parts, one negative and one positive control. protocol. We used 0,2 cm cuvettes. The tubes contained
- 3K
- 6G
- 2M
- 23L
- 18C
- Positive control
- Negative control.
From the seven sampletubes we made 7 LA+AMP plates and 1 LA plate(for the negative control).
Transformation and growth. We made 8 Plates fra each set of transvectorisation (transformation and electroporation), Using this naming scheme: "LA(+AMP) Well# (Electro OR transformation)". For our positive control we have consistently used the commercial [http://en.wikipedia.org/wiki/PUC19 pUC19-plasmid] (a high-copy plasmid containing ampicillin resistance). For the negative control we used competent cells undergoing the same procedure as the transformatants (minus the plasmid addition).
The transformation tubes didn't have any visible pellet after centrifugation in contrast to the electroporation tubes. Also the liquid containing the cells was less opaque for the transformation than the electroporation.
Day two - GROWTH!
Growthcontrol. The transformation plates only showed growth on the negative control without ampicillin. However the electroporation plates showed growth on plates 2M, 6G, 18C and 23L. 18C showed very sparing growth and had a single colony, which we at the time interpreted as an contamination.
It later turned out that J61002 (which J23100=18C sits on) contains an RFP generator and didn't comply to Biobrick standard 10. So we scrapped 18C for a far better construct, namely 13B=J13002, a socalled expression plasmid containing a constitutive promoter(R0040) AND a RBS (B0034). . Also the insert in 3K (p1010=ccdB=Suicide gene) was active, which we hadn't realized prior to this point. Suicide genes properly explain no growth, so we switched to 1K=pSB1A3 with J04450(RFP generator).
Week 3
Day one
Day two
Day three
There was no growth in the backbone ligated with sRIP and RIP. All the controls worked, so the electroporation is working. So we still have problems with either our cuttings or ligations.
We decided to cut each biobrick again.
To test that our enzymes are working, we're going to cut each brick with both enzym 1 and enzym 2, and both enzym 1 and 2. Also we're testing both 30 min and 60 min cutting-time, so for each brick, we're going to have 6 samples. These we're going to run on a gel together with a non-cut brick. Hopefully we'll be able to see how well each biobrick is cut.
We made a new batch of PCR'ed BioBrick, so we have plenty to work with. Tomorrow we are going to run them on a gel, to get a more pure product and then cut that purified product.
After having looked at earlier gels, we concluded that there's isn't alot of DNA in our cut backbone (1K) and decided to pool it all in two eppendorf tube. Hopefully this will give us a better result. (mike)
Day four
Continuing on creating new biobricks from yesterday. Today we ran them on a gel, cut the band that corresponded to the right length, and then purified that gel-piece with a mini prep. After that, we ran 6 different cuts with each biobrick, 2 with the first enzym, 2 with the second enzym and 2 with both enzymes. We then ran one of each for 30 minutes, and another one of each for 1 hour. The idea is to run a small sample of all of them, to compare and check if our cuttings are working correctly. (mike)
Good news! It turns our we have growth on our plates from yesterday after all. We left them for another day at 37 degress celcius, and voila, now there are pretty colonies on the plates. Marc and Julius started the colony-pcr procedure.
