PhD notebook

Cloning newly collected genes of Membranipora and Priapulus. Selected genes and primers:

Membranipora:

Mm PIWIL1 R1 CCGTTGTTGACCTGATGCCACTTTCG
Mm PIWIL1 R2 ACGATGTGATGCTCTCCCTGCTCCATTACC
Mm DDX4 R1 TCTCTGTGTCTTGTCTGGCATACCCATCG
Mm DDX4 R2 CAGTCACCTTTCTCGGGTTTGGACACTCTC
Mm AGO2 F CTTCGCACCTCAGAGGGTAG
Mm AGO2 R CTGGCCCTGAAAGCTACAAG
Mm MEX3 F CCAGTGAGAGGAGCTGAACC
Mm MEX3 R CATCACCACCACACAAGAGG
Mm MAGOH F TCCGCCATTCTTTCTCTTTG
Mm MAGOH R TGGTTGAATGCAAGATGGAA

Priapulus:

Pc PiwiA F GGGTCTTATTCTTTGTTTGC
Pc PiwiA R TTCACGACACCATCGCAG
Pc PiwiB R1 TGCGATACTGCCGACCATCTTCTTGTCC
Pc PiwiB R2 ACGGGATGTGATGGGCTGTAACCTTTCG
Pc Tudor R1 TCCACAAAATAGTAGGCAAGGCAAGGGCTC
Pc Tudor R2 ACGCAACAGATGAACACCTCTACGGCTTGG
Pc Mael F1 CGTGAAAGAGAGCTGGTCCAGTCAGAATGG
Pc Mael F2 CACCGCTTCATAGCTCCAGGTGAAATCC
Pc Mael R1 GCGGTGAAATTCCCTCATAATGCCCTTCTC
Pc Mael R2 TGGACCAGCTCTCTTTCACGCTTATTTCG
Pc Pum F1 ATGAACGCAGTGCCTTGGAACGACTCTCAC
Pc Pum F2 GCAGGACGATGCTATGGTCGGCTATTTC

01/03/2012

Regular and first round of RACE with the above primers.

02/03/2012

Second round of RACE. Run a gel with only 5 µL of each to identify bands to be cut from RACE and direct purify the products of the regular PCR. Ran another gel with the remaining product to cut the bands*. Extracted promising bands and set the ligation reaction at 22:30.

• Did not like this procedure. It is better to run the RACE PCR in 10 comb gel with whole 25 µL to get more signal and extract more.

03/03/2012

Heat-shock transformation and plating. Also ligation for remaining bands (Mm PIWIL1, Pc Tudor 2,3,4, Pc PUM).

04/03/2012

Colony PCR.

05/03/2012

Colony PCR for the remaining bands and repetition for some that there was no positive colonies.

06/03/2012

Plasmid extraction using 1.5 mL of miniprep culture, sequencing reaction, and dropped at the sequencing facility.

08/03/12

Sequences BV25-BV64 returned.