29. Title : gDNA extraction of yll138#

29.1. Date#

17112020

29.2. Objective#

-To have gDNA to make the PCR in order to transform ylic133 and make a bem3\(\Delta\) for SATAY.

29.3. Method#

  • Genomic DNA extraction using the Roboklon KIT

  • I pulled the pellet of 4ml of culture for one extraction.

  • I used in total 4 tubes with 4ml of culture

  • I preheated the elution buffer at 55C before eluting it.

  • I eluted 10ul per tube , so in total I have around 40uL of DNA.

  • Nanodrop concentration: 15ng/uL

    • A260: 0.3

    • A280: 0.1

    • 260/280 : 2.6

    • 260/230: 1.6

  • PCR

    • Protocol Leila (block A machine upstairs)

      • 95C 3mins

      do 35x cycles:

      • 95C 30secs

      • 55C 30secs

      • 72C 1min

      end cycle

      • 72C 5mins

      • 12C on hold

    • 2ul of DNA

    • Primers 51/52 and oLL401/oLL402

    • Adding also 2uL of DNA from yll3a as control.

29.4. Results#

29.5. Next steps#

  • Annealing temperature gradient , with the following T: 50C, 55C , 60C and 65C with 2 mins of extension time. Before was 1 min.

  • Also for the PCR , we used a new dilution of all primers (always vortexing them before diluting.)

  • Using Ramon primers(110/111,114/115), Liedewij primers(oLL401/oLL402) and my primers (51/52).

Amazing!!!

  • New genomic DNA extraction (23112020)

    • 1.5ml of a highly dense culture per eppi

    • 5 DNA extraction per strain (yll138 and yll3a)

    • 30ul of elution

    • concentration on gel (5ul loaded) using BioRad

    Very low DNA concentration and lost of RNA at the bottom

    • yll138 intensity = 0.4 x Intensity (20ng band) = 8ng/5ul ~ 1.5 ng/ul

    • yll3a intensity = 0.2 x Intensity (20ng band) = 4ng/5ul ~ 1 ng/ul

    • These are really low numbers

  • PCR with those genomic DNA and the old DNA that previously worked!

    • Use 5ul DNA template for the new extraction and 1ul of the old yll3a stock

    • Use oLL401/oLL402 and 51/52 primers , newly diluted from the original stock.

    • Use 60C annealing temperature and 2 mins in 72C for the extension.

    • My primers 51 and 52 did not work while Liedewij’s primers did work.

    • Concentration of the PCR product using BioRad

      • Intensity(yll138)= 12.66 * Intensity (20ng)=253.2/5ul=50.64 ng/uL

      • Intensity(yll3a)= 13.43 * Intensity (20ng)=268.6/5ul=53.72 ng/uL

  • One interesting point from this result is that the length of the PCR product from yll138 that have bem3::NAT is the same as the WT. From previous PCR the PCR product size with 51/52 primers is roughly the same as with oLL401/oLL402 in the yll3a (old reference) template.

  • If I have enough PCR product to do both transformations I will continue with it , and check if they acquire NAT resistance , then I will know they are the right clones. Anyways I will send to sequence those clones and I will see what is in there.

29.5.1. Troubleshooting the PCR#

  • Another PCR with 10ul and 1ul from yll138 DNA and 10ul and 1ul from yll3a DNA (old reference) with primers 51 and 52.

    • To test more template and less buffer with the 1ul DNA that may hinder the efficiency of the PCR using yll138 as template.

  • Repeat 8 times the PCR using yll138 as template (5ul) with oLL401/oLL402 to have more PCR product for the transformation.

Confusing! No product for yll138 with oLL primers

29.6. Next Steps#

  • Use a different strain for transformation , this one is causing too much problem and also not giving the right product as it has the same length as the WT, which should not be , given that BEM3 is not there. I will use the one from Wessel that has worked.

29.7. Conclusion#

  • It seems the extension time placed a role in the PCRs

  • I wont continue with this strain (yll138) for transformation.

  • I was overlooking the first step of the genomic extraction kit , which is the activation of the column with Buffer BG. See here the protocol for more details. So this was the reason why I was having so low yield. 😣😣