Title : gDNA extraction from yWT04a

28. Title : gDNA extraction from yWT04a#

28.1. Date#

27112020

To have the DNA template bem3\(\Delta\)::NAT to make the PCR for transformation on ylic133 and ylic136.

Genomic extraction Kit with the Roboclon KIT.
Pellet from 2mL of culture in each eppi. I had 6 eppies.
30uL of elution.
concentration from the Nanodrop: 36ng/uL , much higher than before because of the activation of the column with Buffer BG that previously I was overlooking.

Two PCRs protocol with 1ul and 10ul ywt04a template with oLL401/402 primers using Wessel PCR protocol with 1min in the annealing temperature, instead of 30secs which is how he originally used (no reason). The differences between the two protocols is the extension time, in one is 1min and the other one 2mins.
- 3mins in 98C
- 30X(30s in 98C, 1min in 65C, 1min in 72C)
- 12 mins in 72C
- 4C on hold

{width=50%}

Repeating the PCR with 34X cycles , 10uL template and 2mins 72C extension. (8X) for transformation. (I used the pCR machine from downstairs)
- It did not give any product (super weird!)
- I pulled everything together with the previous PCR and purify it (10uL elution) and I got still very low yield ~8ng/uL.
{width=50%}
Repeat the PCR with the same PCR machine as the one that worked.
- I reduced the time in the annealing temperature from 1min to 30secs. This actually does not make any influence on that. (It did not work - I did not get any bands)
Repeat the genomic extraction (I ran out of DNA)
- I cultured 2 tubes of 10mL to do completely.
{#fig:DNA-extraction-good}

	Nanodrop	On Gel (BioRad)
DNA-ywt04a	60ng/uL	180ng/uL

very high yield of DNA !! :)
PCR using 1uL yll3a , 1uL ywt04a and olic51/52 primers and spy1/spy2 primers from Els as a positive control.
- I changed the annealing temperature to 60C , 30 secs
Do 8 PCRs with 1ul ywt04a template and with olic51/52 primers for transformation.
- PCR protocol: 60C annealing T , 30 secs ; 72C extension 2 mins; 98C denature DNA 3 mins

{width=50%}