Team:KULeuven/7 September 2008

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Lab Work

Wet Lab

Another day, another lab report:

• We did a PCR of the hybrid promoter with the prefix-suffix primers. This gave about the same results as yesterday, albeit a bit better. Maybe because we annealed at a higher temperature this time. Anyhow, we still saw a bit of laddering which we presume to be caused by concatenation of the prefix-suffix primer into constructs like (- Prefix - Promoter - Suffix - )_X. We cut out the (quite broad) band at the right length and extracted the DNA. We hope that by digesting, we once again get the decent cut-out part.
• We made a gel with the PCR samples of the ligation test and with the digests (K145201, K145205, K145253, K145251 and R0040+J23022+J23109+J23032). The result was very good, we now have a lot of new BioBricks that are finished. Only R0040+J23022+J23109+J23032 gave bad results. Both the PCR and the digest was too short. This is the second time that this ligations fails. We think the problem lies in the first part (R0040+J23022). We will not use this one anymore. We wil use R0040+J23066 instead. This ligation definitely succeeded and this part contains key3d and is compatible with the old lock (J23032).
• Yet another failure: PCR of T7 polymerase with UmuD tag failed again (surprise!). We followed the protocol that gave us 3 bands earlier but this time we did only 2 annealing steps at 56 degrees in stead of 3. The third step was replaced by one at 62 followed by 32 at 68 degrees.
• We also found out that the part R1052 doesn't contain a prefix or suffix. That's why the digest and PCR didn't work. We will make some oligos to construct it ourself.
• We electroporated the following parts: K145150+pSB1A2, K145150+E0240, K145150+B0034, K145013+pSB1A2, R0040+P0353, E0040+B0015, B0014+B0033+C0061+B0015 and K145254.
• Set up some ligations: K145201+K145205, K145201+(R0040+E0240), (R0040+B0033)+(C0056+B0015), (R0040+J23066)+(J23109+J23032), (J23109+J23032)+(K145001+B0015) and (J23116+B0032)+(C0062+B0015).

Dry Lab

Designed 2 oligos to construct R0052 (replacing R1052) ourselves. Will enter them as soon as possible.

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