Title : bem1::KanMX transformation in WT+pGal-CDC42

22. Title : bem1::KanMX transformation in WT+pGal-CDC42#

22.1. Date#

06042021-23042021

To have this strain for the growth controls measurements needed for the paper with Fridtjof.
The strain we had yIdb005a,b are completely contaminated with WT. See the last results from 2021-01-25-Supp-controls-sfgFP-influence-to-paper

Yeast Transformation Protocol
06042021: Incubation of ywkd071a in SC-URA+2%Gal+2% Raff
07042021: Culture not in log phase, only at late afternoon , around 15:00. I transfered the culture to room temperature to do the transformation on the next day.
08042021: OD measurement

strain	OD 10x dilution	Real OD	Dilution to OD=0.5	Real OD after dilution
ywkd071a	0.7	~7	10x	0.4

Incubation started at 9:00
Start of transformation at 13:00 , with OD=1.3
10ul of DNA from PCR bem1:KANmx (~200ng/uL) = 2ug DNA
2h recovery step in SC-URA+2% Gal+2% Raff
Plate in selection plates (Sc-URA+2%gal+2%raff +G418)
- 100ul 0x dilution (positive control)
- 100ul 50x dilution (positive control)
- 100ul 100X dilution (positive control)
- 100uL 0X dilution (negative control)
Plate in normal plates (SC-URA+2%gal+2%raff )
- 100uL 100X dilution (positive control)
- 100uL 100X dilution (negative control)

Selection plates {#fig:selection-plates}

Colony PCR with 4 from small to medium size colonies
- Primer sets: olic56/oll60, olic57/oll61, olic56/olic57
- Colonies dissolved in 25ul MiliQ
- 1uL for PCR
- PCR with Leila_LL_60 protocol : 60C annealing temperature, 2min in 72C and 30secs in 98C

Colony PCR Gel {#fig:colony-pcr}

We can not say anything from Figure [@fig:colony-pcr] because it seems the primers oll60/61 dont work. It seems that colony 4 could had a band size for the external primers similar to the positive control, but it did not grow in plate.
Colony PCR of more colonies , one of them a big colony from the 100X selection plate.
- Primer sets: olic56/oll30, olic57/oll29, olic56/olic57
- Colonies dissolved in 10ul MiliQ
- Take 1ul for PCR
- PCR with Leila_LL_60 protocol : 60C annealing temperature, 2min in 72C and 30secs in 98C

Colony PCR with Liedewij primers {#fig:colony-pcr-colony-9}

From Figure [@fig:colony-pcr-colony-9] we can see that colony 9 shows all the exected band sizes. Though it is very vague for the edge selection.

Put all the colonies to grow in SC-URA+2%GAL+2%Raff+G418.
Colony PCR with two more big colonies from the 100x selection plate and two more from the a re-streak plate.

No product for the edges :( {width=50X}

could be because of the colony PCR
19042021-Liquid culture of colonies 10,12,13(they were like dehydrated, not wet colonies in the plate),and 9 , they were the ones that shows growth over-weekend in plates.
- Media: SC-URA+2%Gal+2% Raff+G418
gDNA extraction from these colonies and glycerol stocks
PCR with primers oll29/57 , oll30/56 and olic56/57
Storing three biological replicates for glycerol stocks.Colony 9 did not grow . Strain name : ylic139
- colony 10: a
- colony 12: b
- colony 13: c

PCR with gDNA {#fig:pcr-with-gdna}

Results from the Figure [@fig:pcr-with-gdna]
- replicate ylic139 c shows a weird band size for the upstream and correct integration. So it seems it does not have a correct integration.
- yll3a shows a band for the ptEF terminator , which should not be possible.
- The rest of the replicates seem fine.

Negative controls

Plate the replicates in SC-URA+2% RAff+0% Gal, including yIdb005a (the contaminated stock)
Inoculate them in liquid culture in SC-URA+2% Raff+0% Gal, including yIdb005a (the contaminated stock)

Positive Controls

plate them in SC-URA+2% RAff+2% Gal+G418 including yIdb005a,(the contaminated stock)

Growth of replicates from glycerol stocks in SC-URA+2%Gal+2%Raf+G418.
No growth of yIdb005 in selection media (confirmation)
Miserable growth after 5 days in 0% Gal of the replicates in plates.
Since the glycerols stocks are in 2% Gal they need to be dissolved in 0% Gal to dissolve the Cdc42.

We have two biological replicates of new dbem1:KanMx pGal-cdc42::URA : ylic139 a, b .