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96-well confirmation PCR protocol

Materials and Reagents:

  • Sterile solution basins, 55 ml, Labcor Products, # 730-004.
  • Perkin Elmer 9600 or 9700 PCR machine.
  • Gel Box/combs that work in a 96-well format, Applied Scientific- Shadel.
  • 12-channel pipettes, Brinkmann, 0.5 - 10 µl (#22 45 140-5), 5 - 50 µl (#22 45 150-2), 30 -300 µl (#22 45 160-0).
  • DNA polymerase mix, 20 mM each dNTP, Pharmacia, #27-2094-01.
  • Zymolyase 100T, Seikagaku Corp., #120493-1.

A. Clonal purification

- Pick six G418-resistant colonies from each of the 12 transformations in one row and streak them out to individual colonies on plates containing G418 (200 mg/L). Depending on your personal preferences, between one and six individual isolates can be purified on each plate.

- Incubate at 30 °C for two to three days.

- This is a good time to verify that your heterozygous diploids are met15/MET15. (BY4743 becomes homozygous at MET15 at a reasonable frequency, probably because of recombination at the nearby rDNA region. Heterozygous and homozygoud diploids can be distinguished by their color on lead plates.)

B. Generate a master plate (96-well format)

The master plate is a 15 cm YPD plate containing G418 (200 mg/L) on which 72 individual isolates are arrayed in a 12 by 6 format. The purpose of this step is to get the clonally purified colonies into a 96-well format for the subsequent confirmation PCRs.

Method 1:

Place 25 µl of sterile water into each of the wells of the first 6 rows of a 96-well plate.

Use sterile yellow tips or toothpicks to pick six of the clonally purified colonies into the 96-well plate.

*For example, the six colonies from the first transformation go into wells A1, B1, C1, D1, E1 and F1 respectively. The colonies from the second transformation go into the next column (A2, B2, C2, D2, E2 and F2) and so on.

Use a multi-channel pipette to spot 2.5 µl the water-cell mix onto a large 15 cm petri dish containing G418 (200 mg/L) and incubate at 30 °C for 2 days. Use filter tips to avoid contamination. It also helps to use p10 tips with the multi-channel pipette to make smaller and more uniform spots. These plates should be well dried before you try this. Afterwards, this plate should be wrapped and stored at 4 degrees.

Method 2:

6 colonies purified from each individual transformant can be patched directly into an array on a 15 cm petri dish containing G418 agar in the correct format by taping a microtiter plate underneath the petri dish. Individual colonies can then be picked and patched with a toothpick into a 12 by 6 grid with elements positioned to correspond to the wells in the microtiter dish. If this is done properly, you should be able to stab the plate with a 12-channel pipette, and transfer yeast into microtiter plate wells for further analysis.


Making a frozen backup stock:

Inoculate a 96-well cell culture plate containing 100 µl YPD + 200 mg/L G418 with the cell-water mix that was used to generate the master plate. Alternatively, cells from the master plate can be used to inoculate the cultures (method 2). Incubate at 30°C overnight with mild shaking.

Add 7.5 µl DMSO to the 100 µl cultures and store at - 70°C.

 

C. Confirmation PCR for one Row:

We find it helpful to perform the A-B and A-kanB reactions at the same time. This way the A-B and A-KanB PCR products from one isolate can be run side by side on a gel, making data interpretation easier. The following protocol is designed to check three isolates from 12 different transformations (i.e. one row).

1. Preparation of template DNA


- Add 770 µl of Zymolyase* (300 U/ml) and 3,080 µl water to a tray.

  770 µl              14 µl 300 U/ml Zymo stock
3,080 µl    55 x      56 µl water
--------------------------------------------------------
3,850 µl              70 µl (60 U/ml Zymolyase)


* The 300 U/ml Zymolyase solution is prepared by resuspending 100T Zymolyase in water. The Zymolyase does not go all the way into solution and is stable for several months when stored at 4°C.

Use a multi-channel pipette to transfer 70 µl of this Zymolyase solution to each of the wells in the first three rows A, B, C of a 96-well PCR plate .

Add 20 times the normal amount of cells (a healthy glob, obtained with a loop from a fresh plate) from a wild-type strain to the remaining 1,330 µl of Zymolyase solution in the tray. Use a multi-channel pipette to mix and then transfer 70 µl of this mixture to the wells in row D.

Use a multi-channel pipette to transfer a small amount of cells from the patches on the Master plate into the 70 µl Zymolyase solution.

*Specifically, transfer all of the #1 isolates from the 12 transformations into row A of the PCR plate containing the 70 µl of Zymolyase. The #2 isolates are transferred into row B and the #3 isolates into row C. Row D already contains cells from the wild type control strain.

Incubate the Zymo mixtures at 37 °C for 30 minutes , then 10 minutes at 95 °C (These incubations can be performed in the PCR machine).

Store at 4 °C.

*The zymolyase treated cells can be stored for at least 1 day. We have observed variable results in the confirmations PCRs with cells that were stored for three days




2. ORF Specific Confirmation PCR --> "A-B" primers (upstream junction)

Resuspend the confirmation primers (A, B, C, and D) in 750 µl of TE (~ 10 µM).

Use a multi-channel pipette to transfer 5 µl of the A confirmation primers from an entire row to each of the wells in the first four rows (A-D) of a 96-well PCR plate. Repeat this process for the B confirmation primers. There should be 10 µl of the A-B primers in each of the 48 wells in the top half of the PCR plate.

* Alternatively, you can generate a "A-B" primer mix (enough for four reactions) by transferring 25 µl of the A confirmation primers from one row (e.g. wells A1-A12) to a strip of 12 PCR tubes. Next, transfer 25 µl of the corresponding "B" confirmation primers (e.g. wells A1-A12) to the 12 tubes and mix the 50 µl solution by pipetting up and down. Transfer 10 µl of the "A-B" primer mix to each of the wells in the first 4 rows (A-D) of a 96-well PCR.


3. Transfer template DNA to multiwell PCR plate

Transfer 10 µl of the Zymolyase treated cells into the appropriate wells of the PCR plate containing the 10 µl of the A-B primer mixtures.

*The primer-template solutions should be stored on ice.



4. Prepare and dispense master mix for A-B PCR

Prepare a PCR master mix by combining: 1320 µl water, 275 µl 10x Taq buffer*, 27.5 µl 20mM NTP's, and 27.5 µl Taq Polymerase in a tray on ice.

 
 275 µl        	  5 µl   10x Taq buffer (see below) 
27.5 µl     	0.5 µl   20mM dNTP's (.2 mM)
27.5 µl  55 x 	0.5 µl   2.5 U/µl Taq Polymerase (1.25 U)
1320 µl   		 24 µl   water
        
					 10 µl   Zymolyase treated cells
               	  5 µl   10 µM confirmation primer "A"(1 µM)
               	  5 µl   10 µM confirmation primer "B"(1 µM)

---------------------------------------------------------
               50 µl total volume


*10x Taq buffer contains: 100 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2.


*The final concentrations are shown in parentheses.

Use a multi-channel pipette to transfer 30 µl of the PCR master mix into the wells of the PCR plate that already contain the 20 µl mixture of A-B primers and Zymolyase treated cells.



5. Kan Specific Confirmation PCR -->"A-kanB" primers (upstream junction)

The kan specific "A-kanB" PCR's are performed in the bottom half of the 96-well PCR described above.

Use a 12-channel pipette to transfer 5 µl of the "A" confirmation primers to each of the wells in the bottom half of the 96-well PCR plate (rows E, F, G and H).

* The "kanB" downstream primer will be added directly to the PCR master mix.

Use a 12-channel pipette to transfer10 µl of the Zymolyase treated cells (from step 1) to each of the wells in the bottom half of the plate that already contains 5 µl of "A" confirmation primer.


6. Master mix for A-kanB reaction

Prepare a master PCR mix by combining: 1581 µl water, 275 µl 10x Taq buffer*, 27.5 µl 20mM NTP's, and 27.5 µl Taq Polymerase in a tray on ice.

  275 µl        5 µl   10x Taq buffer* 
 27.5 µl      0.5 µl   20mM dNTP's (0.2 mM)
 27.5 µl      0.5 µl   Taq Polymerase (2.5 U/µl)
          55 x 10 µl   Zymolyase treated cells
                5 µl   10 µM confirmation primer "A"(1µM)
13.75 µl     0.25 µl   100 µM "kanB" primer (1 µM)
 1581 µl    28.75 µl   water
--------------------------------------------------------
               50 µl


*10x Taq buffer contains: 100 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2.


*The final concentrations are shown in parentheses.

Use a multi-channel pipette to transfer 35 µl of this PCR master mix to the bottom half of the PCR plate (rows E-H) containing the 15 µl of "A" confirmation primers and Zymolyase treated cells.


7. PCR: Cycling conditions


The following cycling conditions work well with a Perkin Elmer 9600 and don't work as well with a 9700.

3 min, 94 °C (initial denaturation)

                 --->    15 sec, 94 °C
35 cycles:       --->    15 sec, 57 °C
                 --->    60 sec, 72 °C

3 min, 72 °C (final elongation)


8. Agarose gel electrophoresis

Add 10 µl 6x loading buffer (30% glycerol, 50mM EDTA, 0.25% bromophenol blue) to the 50 µl PCR reactions.

Load 10 µl on a 1.5% agarose gel.

Electrophorese at 100V for 1 hr in the presence of 0.5 µg/ml EtBr.

9. Downstream Junction

Repeat the above protocol for the downstream junction by using the C, D and kanC primers.

see example

last updated February 2002 amchu@cmgm.stanford.edu