Team:UNICAMP-Brazil/Notebooks/September 18

From 2009.igem.org

(Difference between revisions)

Revision as of 02:45, 22 October 2009

Back to Calendar

ColiGuard

PY Promoter - Miniprep and PCR confirmation

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.

Raíssa

@@ Line 21: / Line 21: @@
 ==== CeaB, CeiB and BBa_B0015 restriction, purification and quantification ====
-*<p style=”text-align:justify;”>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 we made DNA quantification. Then, we made DNA purification with kit Pure-link PCR.  </p>
+*<p style=”text-align:justify;”>We made the DNA purification in order to clean all buffer PCR salts.  Immediately, we cut CeaB and CeiB purified with ''Spe''I and ''Xba''I 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 ''Xba''I enzyme restriction and quantificated DNA. Then, we performed DNA purification with Pure-link Purification PCR kit.</p>
 ''Luige''
 ====Cre-Recombinase and pSB1A3 digestion====