Today we performed minipreps (Protocol 2) to extract the plasmids from the selected colonies inoculated yesterday.
After the extraction we performed PCR reactions with 3 pairs of primers to confirm the ligation:
- Ppy-F and Ppy-R
- Ppy-F and VR
- VF and VR
Unfortunately, the sizes of the bands in the agarose gel were not compatible with the expected size of the bands. =(
Fabi and Léo
CeaB, CeiB and BBa_B0015 restriction, purification and quantification
We made the DNA purification in order to clean all buffer PCR salts. Immediately, we cut CeaB and CeiB purified with SpeI and XbaI restriction enzymes. We made that, because we want to join the CeaB DNA with a terminator. The terminator chosen was BBa_B0015. Separately, we cut the terminator plasmid with XbaI enzyme restriction and quantificated DNA. Then, we performed DNA purification with Pure-link Purification PCR kit.
Luige
Cre-Recombinase and pSB1A3 digestion
Today we digested the purified PCR product for Cre-Recombinase without ATG start codon (see Septermber 13th) with XbaI and SpeI restriction enzymes. We also digested the vector pSB1A3 with the same enzymes, aiming in the future ligation between them.
Digestion lasted 3 hours on both cases.
Víctor
YeastGuard
New biobricks - Second screening
This time we tried to find the colonies containing the biobricks by colony PCR (using the forward primer of the insert and the reverse of the plasmid), not digestion. We did 10 reactions of each biobrick. Unfortunately we didn´t find the correct bands.
Taís
We decided to make a bigger screening of our colonies to find the ones containing the biobricks (pJen1, pDLD, Lysozyme, JEN1 orf). We grew 24 colonies from each biobrick O.N. in liquid medium.
"
