Team:UCLA/Notebook/Protein Cages/30 June 2015

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Phillip's notes:

Introduction: Today, a meeting will be held with David in order to check our designed primers for PCquad for pET22b as well as how to design sequencing primers. The order for our gene block and primers will be made when they are done. Today will also be the presentation on PCR by Nithin and Tyler.

Meeting with David: David mentioned that the designed primers for PCquad pET22b looked fine.

Sequencing primers: Between 500-700 bp from each other Two primers within gene for sequencing. Avoid repetitive regions and follow general primer design guidelines. Sequencing primers were designed about 500bp from each other.

We made the following order.

1. 1PCquad Optimized for iGEM 2

Qty: 1 $249.00 Product gBlocks® Gene Fragments 1251-1500 bp Expected Ship Date 7/13/2015 Purification N/A Guaranteed Yield 1000 ngrams

1. 2PCquad primer for pET22b forward

Qty: 1 $5.58 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 10.1 nmol = 95.2 µgrams Length 31 Sequence TCA GAC CAT ATG CCG TTC ATT ACC GTA GGA C

1. 3PCquad primer for pET22b reverse

Qty: 1 $6.66 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 8.6 nmol = 98.3 µgrams Length 37 Sequence TTG ACA GTC TCG AGC TGA CGA TGG CTT CTA TGC TGA G

1. 4PCquad sequencing primer #1

Qty: 1 $3.42 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 15.4 nmol = 90.1 µgrams Length 19 Sequence TGA AAG CCG ATA GAT ATG C

1. 5PCquad sequencing primer #2

Qty: 1 $3.24 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 16.1 nmol = 90 µgrams Length 18 Sequence AAT ACA GAC CTG GAG GTG

1. 6 T7 promoter primer

Qty: 1 $3.78 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 14.2 nmol = 91.5 µgrams Length 21 Sequence TTA ATA CGA CTC ACT ATA GGG

1. 7PCquad amplification primer forward

Qty: 1 $3.60 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 15.7 nmol = 95.6 µgrams Length 20 Sequence ATG CCG TTC ATT ACC GTA GG

1. 8PCquad amplification primer reverse

Qty: 1 $3.78 Product 25 nmole DNA Oligo Expected Ship Date 7/1/2015 Purification Standard Desalting Guaranteed Yield 3 ODs = 15.7 nmol = 101 µgrams Length 21 Sequence CTG ACG ATG GCT TCT ATG CTG

Conclusion: Since the gene block won’t come for another two weeks, the next few days will be focused on preparing for protein expression. The main PCquad papers will be revisited, with special attention to the methods. Once the protocols have been established, the PCquad clone from Yeates will be used to begin the initial protein expression. Perhaps if another part of the team needs assistance, protein expression can be practiced with them.