Brachiopod cloning

Began PCR for cloning Novocrania en, wnt1, wnt5 and Terebratalia fgfr, lfng, pax2/5/8, Fz2, Fz4, Fz5/8, Fz7, Fz10, aldh2, arm.

01/08/13

Gel, Novocrania genes did not work. Others did, extraction and ligation.

2013-08-01 15.09.57

I started a PCR for Novocrania genes using a lower annealing temperature of 55 °C.

02/08/13

Managed one band for engrailed, but not at the right length, slightly shorter.

2013-08-02 18.29.52

03/08/13

I reset the ligation for: lfng, fz58 7, aldh2, arm.

Colony PCR running for the rest.

Also started a PCR with Nano wnt1 and wnt5 with annealing temperature of 50 °C.

05/08/13

Transformed and plated the genes that did not work previously (lfng, fz5/8, fz7, aldh2, arm).

Novocrania gel is not promising, bands are not the right size, need to design new primers.

2013-08-05 13.24.23

Colony PCR went well and today I’ll put the colonies to grow.

2013-08-05 13.24.13

Animal Gene Colony T7
Ttra FGFR 1 BV248
Ttra FGFR 2 BV249
Ttra FGFR 3 BV250
Ttra Pax 2/5/8 1 BV251
Ttra Pax 2/5/8 2 BV252
Ttra Pax 2/5/8 3 BV253
Ttra Fz2 1 BV254
Ttra Fz2 2 BV255
Ttra Fz2 3 BV256
Ttra Fz4 2 BV257
Ttra Fz4 3 BV258
Ttra Fz10 2 BV259
Ttra Fz10 3 BV260
Ttra Fz10 4 BV261
Nano En 3 BV262

06/08/13

Sequences above dropped at the sequencing facility after miniprep extraction and sequencing pcr.

Also, set colony pcr for the genes that hadn’t worked.

07/08/13

Colony gel, putting good colonies to grow today.

2013-08-07 12.43.32

08/08/13

Extracted minipreps for Lfng 3 and 5 and Aldh2 1. Sequencing PCR and dropped samples in the facility.

Gene ID
LFNG c3 BV263
LFNG c5 BV264
ALDH2 c1 BV265