IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/22

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Team Allergy
Today, we are expecting our sequencing results from Genewiz. Once those arrive, we will check the sequences for both the primers and the correct allergen sequence (around 300bps). If the sequences are correct, we will go back to our plasmids and cut the intron out of the plasmid and digest the plasmids containing the sense strands with S, P.

Procedures

 * 1) Check Sequencing Results
 * 2) Digest Intron with X, P
 * 3) Digest Plasmids with Sense with S,P
 * 4) Gel Electrophoresis of Digested Plasmids (both Intron and Sense)
 * 5) Gel Extraction and Purification of Intron and Antisense
 * 6) PCR purification of Sense + Bbone

Results
Sequencing Results
 * Ger S (all 3 samples had correct sequence)
 * Ger A (all 2 samples had correct sequence)
 * Bet 2S (#3 did not work. #2 had correct sequence)
 * Bet 2A (all 3 samples had correct sequence)
 * LTP S (all 3 samples had correct sequence)
 * LTP A (all 2 samples had correct sequence)
 * Pal (#3 did not contain plasmids. #1 and #2 had correct sequence)


 * PME and Bet v1.1 S did not grow up from the plates, so they were not included in sequencing.

Digest Intron with X, P Fast Fermentas protocol was used.

Digest Plasmids with S, P Fast Fermentas protocol was used.

Gel electrophoresis of Digested Plasmids We PCR purified the products of the digestion (unnecessarily), losing some DNA. Elution was done with 50μL of EB. Tomorrow, we will run the purified PCR products on a gel and Gel Extract and Purify.

Colonies from yesterday's ligation
Very few to no colonies.



Sequencing Results
Barstar samples 1, 2, 5, and 6 were confirmed to be correct.

The LacI sequence was confirmed, however we were unable to confirm the presence of the NLS because of its position at the end of the gene where sequencing results are generally unreliable. (NNNNN) Lac1R and Lac3R were poor matches because of what GENEWIZ calls "Early Termination".

Barnase had some serious issues. We are re-growing colonies following ligation and intend to submit some new PCR product as well as the original ADDGENE plasmid for sequencing in the near future.

The Gal4 DNA binding domain samples 1 and 2 were perfect except for a SNP around 320 (in the case of GAL DBD 1) for our sequence and 223 of the biobricks sequence.



Gal4 DBD sample 3 was unsuccessful.

Arabidopsis PCR
FINALLY! EXP2 PROMOTER AMPLIFIED!

~1000kb



Fragments were gel isolated and purified using the QIAgen Gel Purification Kit with the Gel Extraction protocol using a microcentrifuge.


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