RACE PCR for Membranipora and Bugula

03/01/12

Repeating RACE PCR for Bugula genes tried previously since only Bn PUM1 F2 was amplified properly according to the PCR product check. Genes from Membranipora will also be done. Genes and primers (7 reactions):

Bugula Membranipora
Bn PUM1 F1 Mm PIWIL1 R1
Bn PUM1 F2 Mm PIWIL1 R2
Bn PUM1 R1 Mm DDX4 R1
Bn PUM1 R2 Mm DDX4 R2
Bn PIWIL1 R1 Mm MAEL F1
Bn PIWIL1 R2 Mm MAEL F2
Mm MAEL R1
Mm MAEL R2

1st round started at 13:00; 2nd round started at 17:40.

04/01/12

Agarose gel for Bugula neritina:

Bugula RACE Pumilio and Piwi

Only Bn PUM1 F2 had a strong band, the gel was quite similar to the previous RACE PCR run. The faint band marked with “…” Mn PUM1 R2 was cut and frozen if needed in the future.

Gel for Membranipora membranacea:

Membranipora RACE Maelstron, Vasa, Piwi

Mn MAEL F2 had a strong band, as well as Mm DDX4 R2 and Mm PIWIL1 R1. I cut and frozen the short and faint bands Mm MAEL R2 and Mm PIWIL1 R2.

Extraction and ligation done for Bn PUM1 F2, Mm MAEL F2, Mm DDX4 R2, and Mm PIWIL1 R1 at 17:00 and left at 4 °C overnight. Extra pipetting for mixing ligation components.

05/01/12

Heat shock transformation and incubated the 4 plates at 37 °C.

06/01/12

No colonies grew on the plates :( Actually one colony for Bn PUM1 F2. Kevin suggested to do the plating quickly and to not dry out the cells.

I repeated the transformation using the ligation from yesterday, while transforming the regular PCR from Membranipora. Plating at 18:30.

07/01/12

Again the number of colonies was low, only one colony for Bn PUM1 F2 and Mm DDX4 R2. Ligation and transformation needs to be repeated. Started a colony testing for both.

08/01/12

Repeat the ligation for these 4 samples using the ligation control.