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Team:SDU-Denmark/Diary
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Week 1
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 [Team:SDU-Denmark/Protocols/Purification_from_gel|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 [Team:SDU-Denmark/Protocols/Purification_from_gel|protocol] describes.
Day Three - Competence and transformation.
We started the day by making competent cells according to this protocol Team:SDU-Denmark/Protocols/Competent-cells. We measured OD550 (optical density at 550nm wavelength) as:
| #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.
