FGRS: protocol

 

Gene Deletion and Verification

Last Updated

April 3, 2012 1:21 PM


Authored By: Michael Luo, Anna Tseng, Najwa Majida

Edited By: Patrick Killion, Travis White

Protocol: Purify DNA Plasmid Containing KanMX4

Step I: Overnight Culture
In this step you are growing E. coli cells that contain a plasmid with the kanMX4 fragment.


  1. 1.Place 20 mL of LB in 50mL conical tube and add 10 uL of kanamycin.
    Located in white antibiotic box -20°C.

  2. 2.Mix well by inverting tube a few times.

  3. 3.Acquire two small disposable (plastic) E. coli culture tubes with caps.

  4. 4.Allocate 10 mL of LB+kanamycin to each of the two culture tubes.

  5. 5.Use a sterile toothpick to pick a colony of E. coli from plate labeled pFA6-KanMX6.

  6. 6.Inoculate one of the culture tubes.

  7. 7.Repeat steps 4 & 5 for the other culture tube.

  8. 8.Cap the tubes.

  9. 9.Place the two tubes in the incubator in PAI 2.14 (37°C; 180 rpm) to grow overnight.


Note that for an extreme amount of E. coli growth and eventual plasmid purification you may want to grow four rather than two cultures.  The rest of the protocol is based upon the growth of two cultures.  Keep in mind that you can easily grow more.   You should always, however, purify the plasmid DNA on a single column.  See step (5) below for more details.


You may safely stop here.


Step II: Mini-Prep (Also see GenElute Plasmid Mini-Prep Kit Protocol)

In this step you are purifying a stock of the plasmid with the kanMX4 fragment.


  1. 1.Transfer the two 10 mL cultures to two 15 mL conical tubes.

  2. 2.Spin down the 15 mL conical tubes at 6,500 rpm for 5 minutes.

  3. 3.Discard the supernatant.

  4. 4.Re-suspend the cells by adding 100 uL of Resuspension Solution to each 15 mL conical tube.

  5. 5.Transfer the cells to a single new 1.5 mL microcentrifuge tube.

    Steps 6-7 should be done such that one follows the other as quickly as possible.
    Your cells should now be in uL of resuspension solution in one tube.

  6. 6.Lyse the cells by adding 200 uL of Lysis Solution.

  7. 7.Immediately invert the tube gently 6-8 times.
    Now the mixture should look relatively clear.

  8. 8.Add 350 uL of Neutralization Solution. Gently invert the tube 4-6 times.

  9. 9.Pellet the cell debris by centrifuging at 12,000 rpm for 10 mins.

  10. 10.To prepare the column, assemble GenElute plasmid mini spin column into a collection tube; Add 500 uL of the Column Preparation Solution to the mini spin column.

  11. 11.Spin the assembled tube at maximum speed for 1 minute.
    Discard the Column Preparation Solution in the tube below the column.

  12. 12.Transfer the supernatant from step 9 to the prepared column after step 11.

  13. 13.Spin at maximum speed for 1 minute. Discard the liquid in the collection tube.

  14. 14.Add 750 uL of Wash Solution (make sure that EtOH has been added).

  15. 15.Spin at maximum speed for 2 minutes.

  16. 16.Discard the flow-through, and centrifuge again for 2 minutes.

  17. 17.Transfer the column to a new 1.5 mL microcentrifuge tube.

  18. 18.Add 100 uL of Elution Solution directly to the center of the filter without touching it.

  19. 19.Spin at maximum speed for 1 minute.

  20. 20.You now have purified plasmid that contains the kanMX4 fragment.


You may safely stop here.


  1. 21.Run 500 ng of plasmid on a gel to verify integrity of the plasmid.  You should see a distinct and long band.


It should look something like this (courtesy of Mary Alice Neville).




You may safely stop here.


Step III:  PCR

For a description of PCR please see the chapter on Gene Deletion and Verification.


  1. 1.Run a PCR reaction using purified kanMX4 plasmid DNA as the template DNA.
    Primers should be UPTAG and DOWNTAG primers for the gene you are deleting.
    The
    PCR protocol is executed normally.  You should consider running an additional PCR reaction using yeast genomic DNA and ACT1 primers as a positive control.

    You will want to use a PCR program that allows for at least 2000 bp of extension.  I recommend the program 2KB.

    You may safely stop here.

  2. 2.Run 10 uL of the PCR on a 1% agarose gel.


The result should look something like one of the lanes in the image below (Anna Tseng).





Comments on Optimization: For PCR you will likely wish to first setup 50 uL reactions to verify that you have the optimal conditions. A good starting place is a PCR with 50 ng of plasmid DNA template. Simply analyze the reactions on a small agarose gel.  Once you have established the template DNA mass that produces the best result it is then time to scale this up.  You could then setup six matching PCR reactions using master mixing (ask about this).  You would then combine all six PCRs and analyze 5-10 uL of the combined reactions on one lane of a small agarose gel.  If this PCR also shows a good result (single predominant band of the correct size) move onto PCR purification.


  1. 3.PCR purify amplified fragment using a Qiagen PCR purification kit (instructions with kit).

    You may safely stop here.


You now have a DNA fragment with site-specific end-regions containing the kanMX4 fragment.  The site-specific regions will eventually localize the fragment to the correct place in the genome during homologous recombination.


Step IV: Yeast Transformation

  1. 1.Grow a 10 mL overnight culture of yeast to be transformed. 

  2. 2.Measure overnight culture OD.

  3. 3.Calculate amount of overnight needed to create a 25 mL culture of cells at OD 0.5.

  4. 4.Pellet this amount of overnight culture in 15 mL conical tube (3000 rpm, 2 minutes).

  5. 5.Remove media.

  6. 6.Re-suspend cell pellet in 1 mL ddH20.

  7. 7.Transfer to one 1.5 mL microcentrifuge tube.

  8. 8.Pellet at 3000 rpm for 2 minutes.   Remove all traces of ddH20.

  9. 9.Re-suspend in 1 mL 100 mM lithium acetate and incubate 5 minutes at 30°C.

  10. 10.Place 100 µL of cells from the previous step into 1.5 mL microcentrifuge tube.

  11. 11.Pellet cells in microcentrifuge for 5 seconds at 8000 rpm.

  12. 12.Remove supernatant and discard.

  13. 13.Retrieve 20 µL sheared salmon sperm DNA (10 mg / mL).
    Place in microcentrifuge tube.

  14. 14.Boil sheared salmon sperm DNA by placing in 95C heat block for 10 minutes.

  15. 15.Place sheared salmon sperm on ice immediately after 10 minute boil.

  16. 16.On top of yeast cell pellet, add the following things in this order:

    IMPORTANT NOTE: order is important - PEG shields cells from lithium acetate.

    You are NOT adding each of these components and mixing the suspension.

    Do NOT attempt to pipette-mix the components as you add them.

    Instead, you are simply layering the components one-by-one on top of the cell pellet.  The PEG will provide a layer between the cells and the lithium acetate, sheared salmon sperm DNA, transformation DNA and water.

    The mixing will occur after all components are added (during the step 17).

        240 µL polyethylene glycol (PEG) (50% w/v)
        36 µL 1 M lithium acetate
        8 µL sheared salmon sperm DNA (10 mg / mL)
        500 ng to 5 µg DNA (the more the better - I recommend 1000 ng)
        (70 µL - volume of DNA added) sterile ddH20


  1. 17.Vortex mixture at least 2 minutes to suspend transformation mix.

  2. 18.Incubate at 42°C for 20 minutes.

  3. 19.Pellet cells in microcentrifuge for 5 seconds at 8000 rpm.

  4. 20.Remove transformation mixture from top of cells using a micropipette.

  5. 21.Re-suspend cells in 900 µL YPD media. (wash step)

  6. 22.Pellet cells in microcentrifuge for 5 seconds at 8000 rpm.

  7. 23.Remove YPD from top of cells using a micropipette.

  8. 24.Re-suspend cells in 1.3 mL YPD media.

  9. 25.Let cells incubate 2 to 3 hrs to overnight at room temperature on nutator (rocking).

  10. 26.Pellet cells in microcentrifuge for 5 seconds at 8000 rpm.

  11. 27.Gently re-suspend pellet in 500 µL sterile ddH20. (wash step)

  12. 28.Pellet cells in microcentrifuge for 5 seconds at 8000 rpm.

  13. 29.Gently re-suspend pellet in 100 µL sterile ddH20.

  14. 30.Plate suspension on selective media (G418+YPD) using a newly made and sterile culture spreader.  Be sure to spread suspended cells completely over plate.

    Final concentration of G418 in plate media should be 200 µg/mL.


  1. 31.Place plate in 30°C incubator.

  2. 32.Colonies visible in 2-4 days.


You may safely stop here.


You are now growing a plate where each colony represents a single cells that recombined your DNA fragment into the genome.  Thus, this original single cell has resistance to G418.


Step V: Verification of Gene-Deletion Strain


After the plates are grown colonies must be numbered and individually selected for evaluation and confirmation of transformation. (Pick approximately 4-6 colonies).  When picking colonies, it is very important that you leave half of the colony behind in order to maintain a stock that can be used after transformation analysis. Number and circle the colonies on the plate to be able to go back and conduct proper diagnostics of each individual colony.




Confirmation PCR Procedure

  1. 1.Grow an overnight culture of selected colony (or colonies).

  2. 2.Pellet cells in the overnight cultures.

    You may safely stop here.

  3. 3.Perform Genomic DNA Preparation.

    You may safely stop here.

  4. 4.Perform Preliminary PCR Confirmation on Genomic DNA preparation.




Preliminary PCR Confirmation

First you will do a limited set of PCR reactions on candidate colonies.

Reaction 1: Positive Control (ex: ACT1-SHORT-F, ACT1-SHORT-R primers)
Reaction 2: Negative Control (ex: ACT1-SHORT-F, ACT1-SHORT-R primers, no template DNA)
Reaction 3: kanMX4 Interrogation (ex: pKanC and pD where pD is specific to gene deleted).


Complete PCR Confirmation

Reaction 1: Positive Control (ex: ACT1-SHORT-F, ACT1-SHORT-R primers)
Reaction 2: Negative Control (ex: ACT1-SHORT-F, ACT1-SHORT-R primers, no template DNA)
Reaction 3: kanMX4 Interrogation (ex: pKanC and pD where pD is specific to gene deleted).

Reaction 4: kanMX4 Interrogation (ex: pKanB and pA where pA is specific to gene deleted).

Reaction 5: Gene X Interrogation (ex: pA and pB - both primers specific to gene deleted).

Reaction 6: Gene X Interrogation (ex: pC and Pd - both primers specific to gene deleted).


Example PCR Outcomes

In this example the gene was not deleted.


In this example the gene is deleted.

 

Process Overview

  1. Purify DNA plasmid containing kanMX4 fragment by growing E. coli and mini-prepping plasmid DNA.

  2. Use PCR to build DNA fragment you wish to incorporate into the genome.  This fragment must contain a selectable marker.

  3. Transform fragment into live cells.

  4. Allow cells to incubate with fragment - homologous recombination occurs.

  5. Plate cells on selective mediate (YPD+G418).

  6. Allow cells to grow for 48 to 72 hours.

  7. Select and label colonies, grow overnight culture for each colony selected.

  8. Prepare genomic DNA for each overnight, quantitate.

  9. Run PCR reactions to interrogate genomic DNA for existence of original gene, incorporation of kanMX4 fragment.

  10. Analyze PCR reactions by gel electrophoresis.