DNA Protocols

qRT-PCR protocol for B. subtilis (Wang lab)

Adapted from Brigitta and Allison’s protocols by Ponlkrit Yeesin

References: QIAGEN (cat#74106); Invitrogen (cat#18080-051); Bio-Rad (cat#170-8880)

Work Space: RNase free

Cautions: Researcher MUST wear GLOVE at all time, and RNase Zap® may be used to remove RNase from any surfaces as necessary. Sterile aerosol pipette tips MUST be used with RNase-free samples.

Reagent needed

  • Ice-cold 100% methanol (prepare and keep it in – 20 °C  until use)
  • Lysozyme (- 20 °C freezer; need to prepare and use fresh in 1x TE buffer)
  • 1x TE buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA, pH 8)
  • RNeasy Mini Kit (QIAGEN)
  • Buffer RTL+BME (RTL is provided in RNeasy kit but BME is added upon opening RTL)
  • Absolute ethanol
  • DEPC-treated water
  • DNase I (RNase free) Kit (QIAGEN)
  • Random hexamers and reagents for cDNA synthesis are provided in the SuperScript III kit (Invitrogen)
  • 0.1 M NaOH
  • 0.1 M HCl
  • 75% EtOH
  • RNase H (-20°C freezer)
  • iQ™ SYBR® green supermix (-20°C freezer)
  • Primers (see the attached for a guide to design primers for qRT PCR)
  • Double-autoclaved ddH2O (can be used instead of DEPC-treated water)

Materials needed

  • 15 ml conical centrifuge tubes (Falcon™)
  • Double-autoclaved Eppendorf tube (1.5 ml)
  • Aerosol pipet tips
  • Conventional 96-well PCR plates (a drawer under the GelDoc in room 6445)
  • 96-well qPCR plates specific to iCycler iQ system (Bio-Rad)

Note: make sure you use the plate without skirt (clear plate) and “GLOVE” indicated

  • Clear seals for qPCR plates (same place as the 96-well qPCR plates)
  • qPCR machine: iCycler™ Optical Module (Real-Time PCR Detection System) and Software: iCycler iQ™ Optical System Software Version 3.1


There were 3 steps in this protocol. Simply select the proper steps for your experiment.

  1. Sample  collection  and  RNA  isolation (Wang lab UW-Madison)
    1. Streak out strains.
    2. Culture the strains until OD600 ~ 0.200 OR follow step 2a) instead if your cells do not readily/easily grown in liquid medium.
  • Make overnight plates by inoculating two single colonies of each strain into separated Spizizen’s minimal salt solution, and make 1:5 and 1:10 dilutions in Spizizen’s minimal salt solution. Then plate (use glass beads to spread cells) 100μl of each dilution on appropriate plates (2-3 plates per strain).
    1. Incubate all plates at 37 C for 10-12 hour
      note#1: try to get colonies in a log phase – colonies would look very tiny
      note#2: skip this step if growing cells in liquid medium
    2. Collect cells on overnight plates by putting 4-5 ml of S750 minimal media into an overnight plate. Resuspend cells quickly then take cell suspension to other overnight plates (pool 2-3 plates together to get enough cells). Meanwhile, measure OD600 of each cell suspension.
    3. Collect cells by adding cell suspension to 100% ice-cold methanol (kept at -20°C) on ice with 1:1 ratio, i.e. add 5 ml of cell suspension to 5 ml of 100% ice-cold methanol. Rapidly invert samples and put them back on ice.
    4. Spin down samples at 3000g for 15 min at 4 C.
    5. Carefully discard supernatant (excess  methanol), and either freeze down immediately at -80C for future RNA isolation or isolate total RNA from samples  in subsequent steps.
    6. Isolate total RNA from cells using RNeasy Mini Kit (QIAGEN).
    7.  Decant off remaining methanol by inverting tubes over paper towel.
    8. Make fresh 10 mg/ml lysozyme in TE  buffer (10 mM Tris-Cl, pH  7.5; 1mM EDTA, pH 8). Resuspend pellet in 200 μl of lysozyme-TE by vortexing and incubate at 37 C for 20 min.
    9. Add 700 μl buffer RTL+BME (10 μl/ml) and vortex (buffer  RTL  is  provided  in  the kit but BME is added upon opening the new RTL bottle).
    10. Add 500 μl of absolute ethanol and mix well by pipetting up and down.
    11. Load 700 μl of the above mixture at a time on pink columns, and centrifuge for  30 seconds at 8000g. Discard flow through.
    12. Repeat collecting RNA sample on the same pink columns.
    13. Wash the RNeasy spin column (also membrane) with 350 μl of buffer  RW1. Centrifuge for 15 seconds at 8000g. Discard flow through.
    14. Add 10 μl DNAse I stock solution to 140 μl buffer  RDD for each reaction in the master mix (a new Eppendorf tube). Mix gently by inverting the tube 10 times and briefly centrifuge to collect residual liquid (DO NOT VORTEX!).
    15. Add 150 μl of  DNAse I incubation mix directly onto the RNeasy spin column membrane and incubate at room temperature (20-30C) for 15 min.
    16. Add 350 μl buffer RW1 to the pink columns and leave on the benchtop for 5 min.
    17. Close lid gently and centrifuge for 15 seconds at 8000g. Discard flow through.
    18. Transfer pink columns to new 2  ml collection tubes and wash with 500 μl of RPE  buffer. Centrifuge at  8000g for  2  minutes. Discard flow through.
    19. Wash again with 500 μl RPE and spin down at 8000g for 2 minutes. Discard flow through.
    20. Centrifuge at 8000g one more time to dry the column for 1-2 min.
    21. Transfer pink columns to new 1.5 ml tubes. Add 30 μl RNAse-free (DEPC-treated) water directly onto the membrane. Incubate for 5 minutes at room temperature.
    22. Elute RNA by centrifuging at 8000g for 1 minute. Reload the columns with eluted RNA and repeat spinning.
    23. Measure RNA concentration using NanoDrop2000 (Thermo Scientific) and store RNA sample at – 20 C.
  1. First-strand complementary DNA (cDNA) synthesis from total RNA
    1. Combine the following in 1.5 ml microcentrifuge tubes:
  • n μl        1 μg total RNA
  • 1 μl        50 ng/μl random hexamers (provided in the kit)
  • 1 μl        10 mM dNTP mix
  • dilute all the solution with DEPC-treated water to 10 μl
    1. Incubate tubes at 65°C for 5 minutes, then place on ice for 3 minutes. This step is to remove the secondary structures in RNA molecules.
    2. Meanwhile, prepare cDNA synthesis mix by adding each component in the indicated order as following (for 1 reaction):
  • 2 μl         10X RT buffer
  • 4 μl         25 mM MgCl2
  • 2 μl         0.1 M DTT (dithiothreitol)
  • 1 μl         40 U/μl RNase OUTTM (RNase inhibitor)
  • 1 μl        200 U/μl SuperScript® III RT (reverse transcriptase)
    1. Set up the minus RT (-RT; negative control) by excluding reverse transcriptase for each RNA sample
    2. Add 10 μl of cDNA synthesis mix to each RNA/primer mixture. Mix gently and collect the residual fluid at the bottom of the tubes by brief centrifugation.
    3. Incubate tubes as follows (I usually do this step in a thermal cycler):
  • 10 minutes at 25 C
  • 50 minutes at 50 C
  • 5 minutes at 85 C (to stop RT reaction)
  • Chill on ice for 5 minutes
    1. Collect the residual droplets (i.e. at the caps of the tubes) by brief centrifugation. Add 1 μl of RNase H to each tube and incubate at 37 C for 20 minutes (this step is to degrade RNA)
    2. Measure the concentration of cDNA using NanoDrop2000 (Thermo Scientific) and store cDNA samples at -20 C.
  1. Quantification of relative gene expression by real-time PCR (Wang lab UW-Madison)
    1. For each primer pair, set up a master mix for total reaction volume 22 μl as following (for 24 reactions, prepare 30-reaction master mix):
  • 11 μl     SYBR green mix (iQTM SYBR® green supermix)
  • 5 μl     0.25 μM forward primer
  • 5 μl    0.25 μM reverse primer

Note: For an internal control in B. subtilis, our lab has been using accA gene as a reference gene for qPCR normalization (see Livak method). You have to set up reactions for this gene as well as your samples. Fortunately, we have optimal primers, named oJW1221-1222, to amplify the gene. Please keep in mind that you MUST have one internal control for every single qPCR experiment.

  1. Set up the cDNA template mix as the following (for 6 reactions, prepare 10-reaction master mix):
    • m  μl     cDNA sample (to get cDNA sample 100 ng/reaction)
    • n   μl     double distilled water (calculate “n” to reach 10 μl/reaction)
  2. Meanwhile, mix all cDNA samples together in a tube, and this is referred to as “reference pool”. The reference pool will be included in the qPCR reactions (qPCR well #9-11, which contain different amount of total cDNA template (50, 100, and 200 ng, respectively). The purpose of a reference pool is that it is a control of your loading and pipetting as well as the positive and negative effect of PCR efficiency.
    • For how to make a reference pool if one is confused, I would say that you can take 500 ng of each cDNA sample and put them in one tube. Add ddH2O to get the final concentration of 100 ng/μl. Then you can use 0.5, 1, and 2 μl of the reference pool for the cDNA samples of 50, 100, and 200 ng/reaction, respectively.
    • Negative control (-RT) was included in the reaction well #12. (I have one reaction omitted reverse transcriptase in the cDNA synthesis of each sample).
  3. To minimize the variation in qPCR replicates, 2.5X of cDNA samples and SYBR-primer mix were taken and mixed together in a separate conventional PCR plate.
  4. Each reaction will be split into two replicates when the samples were loaded onto a qPCR plate (taken from the plate in section 4).
  5. Three-step qPCR cycles were modified from SYBR-kit manual and performed as the following:
    1. Initial denaturation    95◦C             3 min
    2. 40 cycles of qPCR
    3. Denaturation        95◦C             15 sec
    4. Annealing         55◦C             30 sec
    5. Extension            72◦C             15 sec
    6. Fluorescence data collection

(iCycler iQ™ Real-Time PCR Detection System)

  1. Final extension (optional for subsequent melting curve step)

Note: annealing temperature can be adjusted depending upon your primers

  1. Melting curve was performed after qPCR reaction finished in order to verify the specificity of the amplified products.
  2. From the threshold cycles (CT) acquired from the software in the qPCR machine, relative quantification of gene expression was calculated using the Livak (ΔΔCT) method as described below.
    • Normalize the CT of target gene (i.e. pcrA) to the CT of reference gene (i.e. accA).
    • Normalize the ΔCT of test sample [i.e. Pspac(hy)-pcrA] to the ΔCT of calibrator (i.e. pDR90, empty-vector control)
  3. Calculate the expression ratio, or fold difference using the equation below.
Normalized expression ratio= 2−ΔΔCT

Note: from the equation above, it’s assumed that the primer efficiency (E) is perfect (E=2)

Last modified: 5 August 2021; TBJ

Modified Genomic DNA Extraction (From Dave)


  • Genomic DNA extraction for bacteria including subtilis, C. thermocellum, C. acetobutylicum, E. coli, and Z. mobilis



  • Bacterial colonies (18 hour old)
  • 3-20 mL of rich media, such as LB for subtilis and E. coli or ZRMG for Z. mobilis
  • 540 µL of Lysis buffer
    • 20 mM Tris pH 7.5
    • 50 mM EDTA
    • 100 mM NaCl
  • Lysozyme powder from chicken egg white (Dot Scientific DSL38100)
  • 60 μL of 10% Sarkosyl (N-lauroylsarkosine)
  • 600 μL of buffered phenol
  • 600 μL of 1:1 phenol/chloroform (or 300 μL of phenol and 300 μL of chloroform)
  • 600 μL of chloroform
  • 0 M sodium acetate (~50 μL)
  • 100% Ethanol
  • 70% Ethanol
  • TE solution
    • 12 g Tris (10 mM)
    • 037 g Na2EDTA (1 mM)
    • 1000 ml ddH2O
    • Dissolve and adjust to pH 7.6
  • Nuclease free H2O
  • 5 and 2.0 mL microfuge tubes
  • Test tubes or flasks


  • Stocks of lysis buffer, 10% Sarkosyl, 3.0 M sodium acetate, 70/100% ethanol, TE solution, and nuclease free H2O can be found in the white box labeled Genomic DNA Extraction near Lauren’s bench.
  • Buffered phenol and chloroform can be found in Refrigerator #1. Mix the buffered phenolwhen it’s opened, but not after that. If these solutions have frozen, make sure they have fully thawed before use. Work with these chemicals in a fume hood. Minimize the use/exposure of pipette tips with these solutions because they will melt plastic. Dispense small aliquots of these solutions into a beaker to avoid using a pipette tip in the bottles. When using the buffered phenol, allow the layers to separate. The phenol layer is below the buffer layer.
  • Lysozyme powder is located in the -20⁰C Freezer #2 on the bottom shelf.


  1. Inoculate 3-20 mL of rich media with a fresh colony (18 hours old) in a test tube or flask.
  2. Grow for 3-8 hours with vigorous shaking.
  3. Pellet cells in a 1.5-2.0 mL microfuge tube (1 min/max RPM). Aspirate or pour off the supernatant.
    1. This process can be performed over multiple rounds to pellet more cells. The appropriate amount of culture to pellet is based on the concentration (OD) of the cells. You will need approximately ~3OD*mLs of growing culture (i.e. 3mL of culture at OD=1.0 or 6mL of culture at OD=0.5, etc.)
    2. Cell pellets can be frozen at -20⁰C for future extraction.
  4. Re-suspend the cell pellet in 540 µL of lysis buffer.
  5. Prepare 50 μL of 20 mg/ml lysozyme in lysis buffer.
    1. These lysozyme steps (5-7) are not necessary for coli and Z. mobilis.
    2. Make the lysozyme solution fresh for every DNA extraction. Gently mix the lysozyme pellets in lysis buffer by flicking the tube to mix *DO NOT VORTEX TO MIX*
  6. Add 50 μL lysozyme solution to the microfuge tube.
  7. Incubate the mixture at 37⁰C for 15-30 minutes.
    1. If the cells were harvested at stationary phase, incubate for up to 30 minutes.
  8. Add 60-120 μL of 10% Sarkosyl and vortex.
    1. The suspension should become clear but may still be slightly cloudy.
    2. For subtilis DNA extractions, if solution does not become clear, it is advised you simply start over.
  9. Add 600 μL of buffered phenol and vortex vigorously for 30 seconds.
  10. Spin the mixture at max RPM for 5 minutes.
    1. The aqueous and organic phases should form, with the aqueous phase above the organic phase separated by a white interface (mixture of protein and some DNA).
  11. Carefully collect the aqueous phase into a fresh microfuge tube (~600 μL).
    1. Avoid collecting the white interface, although some carry-over may be inevitable.
  12. Add 600 μL of 1:1 phenol/chloroform and vortex vigorously for 30 seconds.
  13. Spin at max RPM for 5 minutes.
    1. The aqueous phase (top), organic phase (bottom), and white interface should form again.
  14. Carefully collect the aqueous phase into a fresh microfuge tube (~500 μL).
    1. Avoid collecting the white interf
    2. Collecting higher volumes does not result in higher DNA yield.
  15. Add 600 μL of chloroform and vortex vigorously for 30 seconds.
  16. Spin at max RPM for 5 minutes.
    1. The aqueous phase (top), organic phase (bottom), and white interface should form again.
  17. Carefully collect the aqueous phase into a fresh microfuge tube (~450 μL).
    1. Avoid collecting the white interf
  18. Add 1/10 the total volume of 3.0 M sodium acetate (~50 μL) and vortex.
  19. Add 2 volumes of 100% ethanol (~1.0 mL) and gently mix by inverting the tube. Invert the tube until the DNA precipitates as a fluffy white mass.
    1. *DO NOT VORTEX*
    2. If you don’t see a fluffy white mass, it’s okay, continue with the extraction.
  20. Spin at max RPM for 10 Aspirate or pour off the supernatant, leaving only the DNA pellet.
  21. Add ~1.0 ml of 70% ethanol and gently mix.
    1. *DO NOT VORTEX*
  22. Spin at max RPM for 5 minutes. Aspirate or pour off the supernatant, leaving only the DNA pellet.
  23. Repeat steps 21 and 22 an additional time.
  24. Spin the nearly empty microfuge tube at max RPM for 5 minutes.
  25. Carefully remove the supernatant with a p100 or lower pipette.
  26. Air dry or SpeedVac (shared with the Wang lab) the DNA pellet.
    1. To air dry the pellet, place the tube in a biosafety cabinet or fume hood overnight with the cap open.
  27. Re-suspend the DNA pellet in 30-100 μL TE solution or ddH2O.
    1. The DNA pellet may take several hours or overnight to fully dissolve.
  28. Store the DNA solutions at -20⁰C.

Last modified: 09 August 2021; LNL

TBE electrophoresis buffer preparation (10X)

Reagent Quantity Final concentration
Tris base 121.1g 1M
Boric acid 61.8g 1M
EDTA (disodium salt) 7.4g 0.02M

Bring to 1L with ddH­2O water. Store at room temperature for up to 6 months. Dilute 100mL to 1L to make 1X gel running buffer. If you are making or diluting a large amount of buffer, get water from the core instead of using the water from the carboy Dave fills.

If there is precipitation at the bottom of the 10X stock, rinse the entire jug and start over.

Notes: In the lab, we have one 3L jug for 10X TBE. Separately, we have a 10 L jug for 1X TBE. Make 3L of 10X at a time and use it to refill and dilute in the 10 L jug. Note the dates on the jugs for when you’ve remade the 10X or diluted to 1X. If there’s precipitate in either solution, toss and remake.

Last modified: 5 August 2021; TBJ

Last modified: 09 August 2021; LNL

PCR transformation protocol for B. subtilis


Day 0 – Preparation

  • Order two sets of primers:
    1. One set that will amplify a 2-2.5 kb region from the B. subtilis 168 background for the transformation and
    2. One set that will amplify a shorter chunk, from just outside the gene or antibiotic resistance gene in the NCIB 3610 background for confirmation of a successful transformation.
    3. For the larger chunk, go on http://subtiwiki.uni-goettingen.de/ and type in your gene of interest. Alternatively, Benchling can be used if you have the B. subtilis 3610 genome. I am going to provide an example:
      1. Type in “fapR” in the search bar and press enter. (same for Benchling).
      2. You should see a fapR page with information about the gene, including the bp sequence. Click on the blue “Sequence” link. (In Benchling, your gene of interest will be highlighted. Select a 2.5 KB region surrounding your gene of interest. Skip to step iv. Copy/paste into IDT primer design.)
  • Now you should be at a page titled “Genome Browser”. Click on the tab called “Gene with flanking regions”. Enter the gene name. Because the kanR cassette is 795 bp long, we want to get primers for 2.5kb – .8kb = 1.7kb / 2 = .85 kb flanking the gene on each side. Type 850 bp for upstream and downstream of the gene. Scroll to the bottom and there’s your sequence.
  1. Go to https://www.idtdna.com/PrimerQuest/Home/Index. Make an account if you don’t have one and then log in. Paste the full sequence in where it says ‘Sequence Entry’ and click the grey box that says “show custom design parameters”. Leave the default “Task Settings”. Go down to “Primer Criteria” and modify the Primer Tm to what you want. I have had optimal success at 53C, so I set the range from 51-55C. Then adjust the amplicon maximum to allow your entire sequence to be amplified. And where it says “Excluded Region List”, enter values so that primers are created for the ends of your amplicon. In my fapR example, the length of the amplicon says 1 to 2267, so I am going to exclude bp’s 200-2000. Select “Get Assays”. Download assays and organize excel sheet so Dave can order primers for you. One column with the primer names and one column with the sequences.
  1. For the smaller chunk, go on Benchling and look up your gene in the B. subtilis 3610 genome. Go about 100bp outside of the gene upstream and downstream and copy/paste into IDT primerquest again. Use the same default parameters, but adjust the excluded region to exclude the first and last 100 bp’s. For fapR, it’s 100-665. Select “Get Assays”. Download assays and organize excel sheet so Dave can order primers for you. One column with the primer names and one column with the sequences.
  • Prepare 2.5 kb PCR product:
    1. If you haven’t already, streak out your desired mutant 168 strain from the strain collection in the -80C. There is an excel sheet on the server with all the strain locations. To take a strain from the collection, sterilize a razor blade and cut a square out of the sealed plate for only the well that you need. Using a new, sterile plate cover, cut a small piece out, and re-cover the well aseptically. This strain collection has kanamycin resistance, so streak on LB + kan plates. Incubate overnight in 37°C room or incubator. The next morning, grow a culture for DNA extraction and grow a culture to make more easily accessible freezer stocks.
    2. Do a DNA extraction on your mutant 168 pellet. And use this gDNA as a template for your 2.5 kb PCR amplification.
    3. Follow PCR protocol for amplifying a 2.5kb region of the desired mutant strain of B. subtilis 168. Do this PCR in 22 tubes, leaving the last two tubes for WT 3610 and a no template well as your control. It is important to do this amplification in many wells because they will all need to be concentrated for the transformation.
    4. After your PCR, run a gel. Because we are amplifying a large chunk of DNA, it may be difficult to distinguish between WT and 168 PCR products. Run your gel longer if you need to or sequence your amplicon to ensure the kan resistance cassette is present.
    5. Do a PCR cleanup using a QIAquick PCR purification kit. To concentrate your PCR product, simply load all PCR product tubes onto the same Spin column in the purification protocol. About 6 wells of PCR product fit in the spin column at once. Additionally, use 50 uL of DNA/RNAase free PCR water to elute. Let water sit on filter for one minute before centrifuging. Optional step: After centrifugation, suck up purified DNA fragment and reapply it to the same filter. Let sit for one minute and centrifuge again.
    6. Quantify PCR product using Nanodrop in core. Aiming for ~500 ng/uL. More is better. You want a total of 1500-2500 ng to mix in your tube with competent cells.
  • Prepare LB media and plates with the appropriate antibiotic (erm or kan; check your sequence for antibiotic genes).
  • Sterilize “hockey stick”, rounded-bottom glass rods, or beads (choose whatever you have or whatever you are comfortable using).
  • Sterilize tubes (small test tubes with yellow lids or eppendorfs).
  • Make 10X MC Media (competency media): https://docs.google.com/document/d/1USu-RptmucLvrO7NcybNfYyWxTs6g6RapJsiFK9y5iU/edit?usp=sharing
Component For 100 ml For 200 ml For 500 ml
K2HPO4 (potassium phosphate dibasic) 10.7 g 21.4 g 53.6 g
KH2PO4 (potassium phosphate monobasic) 5.2 g 10.5 g 26.2 g
Glucose (dextrose) 20 g 40 g 100 g
Na3C6H5O7 * 2H2O (Sodium Citrate Dihydrate) 0.88 g 1.8 g 4.4 g
1000x Ferric Ammonium Citrate

(2.2 g Ferric Ammonium Citrate per 100 ml ddH2O *light sensitive compound*)

1.0 ml 2.0 ml 5.0 ml
Casein Hydrolysate (oxoid) 1.0 g 2.0 g 5.0 g
Potassium Glutamate monohydrate (L-glutamic acid) 2.2 g 4.4 g 11 g
ddH2O To 100 ml To 200 ml To 500 ml

Day 1

  1. Plate WT subtilis 3610 on LB plate (no antibiotics) and grow overnight at 37 °C  (no longer than 18 hrs).

Day 2

  1. Dilute to 1X MC: 9 mL DI water + 1 mL 10X MC + 33 ul 1 M MgSO4. With 10 mL of 1X MC, make 3 tubes of media with 3 mL each to be inoculated. Make less if you need less, more if you need more.
  2. Pick a colony and inoculate in 1X MC media (volume of MC depends on how many transformations you are doing; 3 mL of 1X MC per tube and 200 uL of culture is required per one transformation).
  3. Measure OD600 every 30 minutes after 3-4 hours of growth. Ideally, stationary phase (OD600 remains constant across 2 time points) should be reached by 4-4.5 hours.
  4. Once stationary phase is reached for all sets, proceed with transformation.
  5. Aliquot 200 uL of the stationary culture to culture tubes
  6. Add DNA product of choices to the aliquot stationary culture in each tube.
    1. 1500-2500 ng of PCR product per transforming culture will work best.
  • If you have 100 ng/ul PCR product, you need to add 15 ul of the PCR product to your 200ul stationary culture. Higher concentrations and lower volumes will work better.
    1. Shake tubes with stationary culture+DNA product at 37 °C
  1. PCR product: shake for 5 hours
  • Take antibiotic plates out of fridge to warm up at room temp subtilis are sensitive to low temperature
  • Plate all the incubated culture onto the warm antibiotic plate
    • . use either sterile “hockey stick”, rounded-bottom glass rods, or beads to streak the culture until most of the liquid has dried.
  1. Optional: you can try diluting down the culture before plating.
  • Grow the plates overnight in 37 °C (no more than 18 hrs).

Day 3

  1. Re-streak single colonies to get another round of single colonies (4 to an antibiotic plate) and grow overnight at 37°C.
  2. If no colony has grown at all, double the amount of DNA product or increase the incubation time with the stationary culture by 1 hour.

Day 4

  1. Pick individual colony the grown on the re-streak antibiotic plates and inoculate them individually in 3 ml LB (optional antibiotics).
  2. Grow at 37°C & shake well for at least 3 hours.
  3. Collect the culture for DNA extraction (for later confirmation of transformation by PCR) or mix with 50% glycerol at 1:1 ratio to make frozen culture for -80°C storage.

PCR confirmation of insert:

  1. Use primers to amplify your gene of interest. These primers should start ~100bp outside your gene so that you can compare WT to the mutant strain. The kan cassette is ~750 bp.


    1. Order Primers for the antibiotic resistance gene itself.
      1. If the wild type gene and resistance gene are close to the same size, you will need to PCR out of the resistance gene to the WT backbone.