Chewbby

PCR Fusion (SOEing) protocol

 

Step 1: Amplify separately both fragments to be fused together

2 PCR mixes (final volume 50 µl):

    Volume (µL)
Buffer 5X 10
dNTP 2.5 mM each 5
Forward Primer 10µM 2,5
Reverse Primer 10µM 2,5
Phusion 0,5
DNA template 10 ng 1
H20 (qsp 50µL) 28,5

Set of primer (stock solution 100 µM used at 10µM)

    PCR 1 (left PCR) PCR 2 (right PCR)
Primer Forward :    
Primer Reverse :    
Expected size :    
DNA template (genomic or plasmid):    

PCR plan :

  1 2
A PCR 1 PCR 2

PCR Program :

Temp. Time  
98 °C 5 min  
98 °C 20 sec X35
56 °C 20 sec
72 °C 2min30sec
72 °C 3 min  
4 °C  

 

Run 5 μL of PCR products in a 1% agarose gel to verify products.

For all samples that gave a band of expected size, purify the remaining PCR products using Macherey-Nagel PCR clean-up kit.

Measure DNA concentration with Qubit (BR kit)

Mix PCR product of PCR1 and 2

Mix ~250 nmol of left and right PCR products in a final volume of 8 µl.

The formula to go from ng to nmol is: ng x 106 / (660 x size in bp) = nmol

      Size (bp) concentr. (ng/µl) nmoles DNA Volume (µL) for 250 nmoles H20 Volume (µL) for 250 nmoles
  PCR product PCR 1          
  PCR product PCR 2          

Step 2: PCR SOEing

PCR mix (final volume 25 µl):

    Volume (µL)
Buffer 5X 5
dNTP 2.5 mM each 2,5
MgCl2 2
Phusion 0,25
DNA mix 8
H20 (qsp 25µL) 7,25

DNA mix: 8 µl of ~250 nmol of left and right PCR products

PCR plan :

  1
A Mix PCR 1+2

PCR Program :

Temp. Time  
98 °C 5 min  
98 °C 20 sec X25
56 °C 20 sec
72 °C 2min30sec
72 °C 3 min  
4 °C  

 

Step 3: amplifying SOEing product (or SOEing step 2)

PCR mix (final volume 50 µl):

    Volume (µL)
Buffer 5X 10
dNTP 2.5 mM each 5
Forward Primer 10µM 2,5
Reverse Primer 10µM 2,5
MgCl2 4
Phusion 0,5
DNA template 1
H20 (qsp 50µL) 24,5

DNA template : 1ul of previous PCR product (step 2)

PCR plan :

  1
A Mix PCR

PCR Program :

Temp. Time  
98 °C 5 min  
98 °C 20 sec X35
56 °C 20 sec
72 °C 2min30sec
72 °C 3 min  
4 °C  

Run 5 μL of PCR products  of step 2 and step 3 in a 1% agarose gel to verify products : there could be a smear of larger products.

Do NOT clean up afterwards, to avoid losing too much product. Use within 48 hours for transformation.

Transformation in Yeast

Do not forget to thaw yeast strains to be transformed and to prepare selection plates and reagents a few days before transformation.

  Strains :
  nb of dilution

Do not forget to turn on 42°C water bath before step 9C.

A. Grow the yeast strain(s) to be transformed in 5 mL of YPD at 30°C overnight. If possible, it is best to start cultures from a patch on a YPG plate (5% glycerol).

B. Dilute 200 μL of overnight culture into 5 mL of YPD and grow for an additional 4 hours at 30°C.

C. Pellet cells by centrifuging at 3000 rpm for 5 minutes and boiled ssDNA at 102°C during 10 min aproximately (use it a TA)

D. Resuspend in 1 mL sterile H2O and transfer to a micro-centrifuge tube.

E. Centrifuge for 1 min at 11,000 rpm and remove supernatant.

F. Resuspend in 0.1 M LiAc, centrifuge for 1 min at 11,000 rpm and remove supernatant.

G. Combine (or not if not mutch samples) > 500 ng of plasmid (pGY638 (pGuid Lys2)) with > 1 μg of repair fragment  (PCR fusion product) to a final volume of 34 μL (usually 2 μL of plasmid + 32 μL of PCR product).

  Transfo name plasmid Repair Fragment Yeast Strain
       

Do step H in less than 5 min / do not make more than 6/4 samples in the same time / respect the deposit order

H. Add to the cells 240 μL 50% PEG, 36 μL 1 M LiAc, 50 μL boiled Salmon sperm DNA (2 mg/mL) and 34 μL of plasmid / repair fragment mixture.

I. Immediately vortex thoroughly until the pellet is dissolved. The pellet becomes difficult to resuspend after ~5 min in 50% PEG

J. Incubate at 30°C (in incubator) for 30 min, followed by 42°C for 30 min in water bath.

K. Centrifuge at 11,000 rpm for 1 min and remove supernatant.

L. Resuspend in 1 mL sterile H2O (be gentle, the pellet can be difficult to resuspend at this step, so take your time).

M.Centrifuge at 11,000 rpm for 1 min, remove supernatant and resuspend in 110 μL sterile H2O.

N. Plate 100 μL on SD-Ura medium (pur). Add 90 μL sterile H2O to leftover and plate remaining 100 μL on a second SD-Ura plate (1/10)

O. Grow at 30°C for 2 days (or keep on the bench if you cannot take care of the plates in the next 3 days).

Validation of transformants

A. Streak several colonies for singles on fresh SD-Ura plates and grow at 30°C for 2 days. Up to 8 colonies can be streaked on the same plate. The number of colonies to be streaked depends on the expected rate of false positives (inversely correlated with the total number of colonies obtained). It is recommended to streak at least 8 colonies per transformation and up to 24 colonies for tricky transformation (eg. Insertions > 3 kb).

B. Patch one colony from each streak on a YPG plate (5% glycerol) and grow at 30°C for 2 days. Up to 20 colonies can be patched

C. Amplify the targeted genomic site by PCR using primers designed for validation as described below.

PCR master mix :

      Volume (µL)
One Taq Quick load 2X Master Mix with Standard buffer 12,5
Primer Forward (10uM) 0,5
Primer Reverse (10uM) 0,5
H20 (qsp 25µL) 11,5

PCR plan :

  1 2 3 4 5 6 7 8
A                
B                

D. From each patch that grew one YPG, take an amount of yeast corresponding to the size of a colony with a pipet tip and crush the cells in an empty PCR well (use PCR strips or 96-well PCR plates depending on the number of samples).

E. Add 25 μL of PCR mix in each well. For each pair of primers, always include the following controls: one well without cells and one well with cells from the progenitor strain used in step 9A.

PCR Program :

Temp. Time  
94 °C 2 min  
94 °C 30 sec X30
55 °C (or Tm-5°C) 30 sec
68 °C 3min (1min/kb)
68 °C 5 min  
4 °C  

F. Run 5 μL of PCR products in a 1% agarose gel.

G. For all samples that gave a band of expected size, purify the remaining PCR products using Macherey-Nagel PCR clean-up kit.

H. Send for sequencing with eurofins suprem Run Tube.

Remove plasmid

A. For clones with correct sequence (check for point mutations), patch cells from YPG plate on 5-FOA plate. Grow for one day at 30°C.

B. Grow cells from 5-FOA patch into 5 mL of YPD at 30°C overnight.

C. Glycerol stock 1 mL of culture and keep in GY -80°C collection.