One completely random project I worked on was trying to extract DNA from silkworm for Next-Gen Sequencing. This proved to be harder than I imagined from what I hypothesize to be endogenous DNases (https://www.tandfonline.com/doi/abs/10.1080/00021369.1972.10860497 ). An agent to inactivate them, or perhaps harvesting the silkworm at the time of sacrifice would stop this from occurring (These ones were frozen in liquid nitrogen originally). In essence, this experiment was a failure!

Initially, the Silkworm was processed using the Qiagen DNA Mini Kit [51304] (Lane 1) and gave low yield and DNA that we suspected was sheared or degraded. 

We then attempted to use the Qiagen DNA Mini Kit [51304] (Lanes 2 and 3), with differing homogenization strengths in order to see if that was the issue. Ultimately it gave a low yield, but showed a similar degradation pattern as before.

Suspecting it may have been an issue with the Qiagen kit and its columns, we switched to the more robust Epicenter kit [MCD85201] (Lanes 4 and 5), and determined that DTT might improve the yield due to its ability to help extract DNA from the chitin so we decided to incorporate that as well. We also switched to using a mortar and pestle as a gentler method in comparison to the tissue ruptor. 

Additionally, we processed 2g of the Silkworm with a mortar and pestle, suspended it in 4800uL of Lysis solution from Epicenter with 0.1M DTT and Proteinase K, and left it to lyse overnight at 55C. The following day we processed it using the Epicenter kit (MCD85201) and it had no yield whatsoever. This result is not present on the E-gel.

Due to the aforementioned result, we did some more research and discovered that silkworms have DNases that are present so that could be the primary cause of degradation and low yield. We used a custom lysis buffer that is high in salt and proteinase K in order to combat this issue. The 30mg of silkworm was processed quickly and lightly with the tissue ruptor in the lysis solution while still cold, thus hopefully preventing the activation of any DNases. Ultimately, we still saw degradation (Lane 6).

Lastly we decided to do a combination of the custom lysis buffer with the protein precipitation step from Epicenter since there was a lot of fragmented material and residue when we performed the extraction before. This did not alleviate the problem either. However, it seemed to improve the yield (Lane 7).

Conclusion: DNA degradation is occurring with the silkworm possibly as a result of endogenous DNases present in the silkworm (https://www.tandfonline.com/doi/abs/10.1080/00021369.1972.10860497 ). Perhaps extraction of gDNA from the silkworm has to occur at the moment of harvesting or shortly thereafter. 

Figure: Silkworm genomic DNA extractions using Qiagen, Epicentre and Custom Lysis methods. 5uL of each sample was loaded on the gel.
Lane L: GeneRuler 1KB Plus DNA Ladder
Lane1: gDNA extraction using the Qiagen DNA Mini kit (51304), tissue ruptor was used to homogenize 100mg of Silkworm. Final elution volume of 100uL
Lane2: gDNA Extraction using the Qiagen DNA Mini kit (51304), tissue ruptor was used to homogenize 30mg of silkworm. Weak tissue homogenization was performed. Final elution volume of 100uL.
Lane3: gDNA Extraction using the Qiagen DNA Mini Kit (51304), tissue ruptor was used to homogenize 30mg of silkworm. Heavy tissue homogenization was performed. Final elution volume of 100 uL.
Lane4: gDNA Extraction using Epicenter (MCD85201) with 0.1M DTT added to the lysis solution. Homogenization with mortar and pestle was performed on 30mg of silkworm. Final elution volume of 50 uL.
Lane5: gDNA Extraction using Epicenter (MCD85201) without DTT. Homogenization with Mortar and Pestle was performed on 30mg of silkworm. Final elution volume of 50 uL.
Lane6: gDNA extraction using high salt custom lysis buffer, B-mercaptoethanol. Homogenization using tissue ruptor was performed on 30mg of silkworm. Final elution volume of 100 uL.
Lane7: gDNA Extraction using custom lysis buffer from adjacent lab followed by Epicenter kit (MCD85201) for Protein precipitation.  Homogenization using tissue ruptor was performed on 30mg of Silkworm. Final elution volume of 100 uL.



Extracting DNA from Silkworm is Difficult potentially due to endogenous DNAses

Extracting good quality mtDNA from human hair is tough. It took me months of testing tens of different protocols, wash concentrations and methods to be able to get clean extractions with low amounts of contamination. Hair is super tough, due to the keratin fibers that exist there, and to get mtDNA out, you actually have to break it down (the mtDNA circles are trapped inside). The DNA part is actually pretty easy (just get the hair at the root). It’s a meticulous process, and you need a LOT of starting material. MtDNA exists in such small quantities that you need quite a bit to even get 1 ng of it. Subsequently, if you’re doing any next-gen sequencing, using a library preparation kit that works with low input amounts (i.e Nextera) will be our best bet. I created a small protocol to help with the wash and extraction procedure. The protocol is adapted from this paper (Protocol adapted from : Assessing Deep Sequencing Technology for Human Forensic Mitochondrial DNA Analysis,  Mark R. Wilson. Ph.D.)

 

EXPANDED PROTOCOL

Clean up:

Obtain 2cm of hair shaft (get a few of these pieces). For 1ng need around 100+ 2cm pieces.

Make 5% terg-a-zyme mixture (i.e: 2g in 40 mL)

suspend hair in 5% terg-a-zyme (1 mL) in an eppendorf tube, place in incubator at 37˚C , 900 rpm for 20 minutes. 

Remove the 1 mL of 5% terg-a-zyme and add 1 mL of nuclease-free H2O, incubate for 10 minutes. Flick the tube every 2-3 minutes.

Remove 1mL of H2O, and add 100% ethanol. Incubate for 10 minutes. Flick the tube every 2-3 minutes.

Remove the 100% ethanol and let the hair dry. Use a P10 or P20 to remove any remaining drops. All ethanol must be removed.

Extraction:

Place clean hair fragment/s in a solution of 300 uL Qiagen Buffer ATL, 20 uL 0.6 U/uL proteinase K (12U of prot. K) and 40 uL of 0.5M DTT. 6 ul of 50mg/ml proteinase K. for 100 hair pieces, add 600 uL buffer atl, 12 ul of proteinase K and 80 uL of 0.5M DTT.

Digest for 1-2 hour at 56˚C, 900 rpm on a thermal shaker.

Add 300 uL of Qiagen Buffer AL to the digestion solution and incubate at 70˚C, 900 rpm for 10 minutes. Add 600 uL for 100 hair pieces.

4)Proceed to the magnetic bead step (addition of 15 uL prepfiler magnetic particles) in the Prepfiler forensic DNA extraction Protocol.PrepFiler Kit

Vortex the PrepFiler® Magnetic Particles tube for approximately 5 seconds, invert the tube to confirm that no visible pellet remains in the bottom of the tube, then centrifuge briefly. For multiple samples, vortex the magnetic particles tube every 5 minutes during the next step.

Pipet 15 µL of magnetic particles into the tube containing the sample lysate.

Cap the sample lysate tube, vortex it at low speed (approximately 500 – 1200 rpm) for 10 seconds, then centrifuge it briefly.

After centrifuging, add 180 µL of isopropanol to the sample lysate tube

Cap the sample lysate tube, vortex it at low speed (approximately 500 – 1200 rpm) for 5 seconds, then centrifuge it briefly

Put the sample lysate tube in a shaker or on a vortexer, then mix at room temperature at 1000 rpm for 10 minutes

7)Vortex the sample DNA tube

*If magnetic particles are present on the sides of the sample DNA tube above the meniscus, invert the tube to resuspend the particles.

*Vortex the sample DNA tube at maximum speed (approximately 10,000 rpm) for 10 seconds, then centrifuge briefly. 

Note: It is acceptable to have magnetic particle aggregates suspended in the solution or on the side of the tube below the meniscus

10) Place the sample DNA tube in the magnetic stand and wait until the size of the pellet of magnetic particles on the back of the tube stops increasing (approximately 1 – 2 minutes). 
11) With the sample DNA tube remaining in the magnetic stand, use a pipette to carefully remove and discard all visible liquid phases. Do not aspirate magnetic particles or disturb the magnetic particle pellet. You can use a size P200 or P1000 pipettor to remove most of the liquid, then use a size P20 pipettor to remove the remaining liquid.


With the sample DNA tube remaining in the magnetic stand, open the tube, then allow the magnetic particles-bound DNA to air-dry for 7–10 minutes (air-drying for more than 10 minutes may reduce DNA yield). If the room temperature is >25°C, reduce drying time to 5 minutes.

Bring the thermal shaker temperature to 70°C.

Add 50 µL of PrepFiler® Elution Buffer to the sample DNA tube. Do not use water. You can use low-TE buffer.

Cap the sample DNA tube, vortex it at maximum speed (approximately 10,000 rpm) until there is no visible magnetic particle pellet on the side of the tube (approximately 5 seconds), then centrifuge it briefly

Place the sample DNA tube in a thermal shaker (or heat block), then incubate at 70°C and 900 rpm for 5 minutes. If you use a heat block, briefly vortex and centrifuge the tube every 2 – 3 minutes.

Vortex the sample DNA tube at maximum speed (approximately 10,000 rpm) until there is no visible magnetic particle pellet on the side of the tube (approximately 2 seconds), then centrifuge briefly

Place the sample DNA tube in the magnetic stand, then wait until the size of the pellet at the side of the tube stops increasing (at least 1 minute).

Pipet the liquid in the sample DNA tube (which contains the isolated genomic DNA) to a new spin tube or 1.5-mL microcentrifuge tube for storage. Do not aspirate magnetic particles or disturb the magnetic particle pellet.

If the eluted DNA extract is turbid, centrifuge the tube for 5 – 7 minutes at maximum speed (approximately 10,000 rpm), then transfer the clear supernatant to a new 1.5-mL microcentrifuge tube.

The isolated DNA can be stored at 4°C for up to one week, or at –20°C for longer storage.
 
Check the sample using mitochondrial genome selecting primers and ones for some NUMT sequence. That is, run a PCR reaction with a set of mitochondrial primers on one amount, and nuclear primers on another amount. The nuclear primer is to check for nuclear DNA contamination, and the mtDNA set is to see if you have mtDNA Present. 

Lane 1: Optimized Qiagen/Prepfiler extraction on hairsample1 (20 2cm pieces); nuclearprimer1 and mtprimer1 30 cycles, loaded 9uL x2 on gel
Lane 2: Optimized Qiagen/Prepfiler extraction on hairsample1 (100 2cm pieces); nuclearprimer1 and mtprimer1  30 cycles, loaded 9uL x2 on gel
Lane 3: PCR negative control nuclearprimer1 and mtprimer1  30 cycles, loaded 9uL x2 on gel

Note: The lower band is the nuclear primer and the upper band is the mtDNA one, the lowermost band is just runoff primers.

mtDNA extraction and wash from Human Hair

This protocol runs several in vitro Cas9 cleavage conditions for identifying the optimal setup for a specific target (in this case the Sub1 gene).

Cas9 Cleavage

1. Cas9 nuclease reactions can be set up as described in the table below. For identifying cleavage conditions, use a combination of 1X and 2X Cas9 reactions along with no sgRNA controls. See Table 1.

2. Incubate the reaction mix for 10 minutes at 37 C.

3. Add 4 ul of DNA substrate.

4. Incubate the reaction for 2 hours at 37 C.

5. Denature the Cas9 nuclease with a 10 minute incubation at 80 C. If necessary, perform a chloroform extraction on the digestion products.

Chloroform Extraction

1. Aliquot 1 ml chloroform to use as a stock solution.

2. Add 1 volume of chloroform to the microfuge tube containing Cas9 reaction solution. Gently pipette up and down 10 times.

3. Centrifuge the tube and aliquot the top layer to a new microfuge tube. This solution may be used for electrophoresis.

4. Leave the tube of stock solution chloroform in the hood until the chloroform evaporates. 

MetaPhor Agarose Gel Electrophoresis

1. Prepare a 3% agarose gel with the following components in a 1 L flask:
- 5 g MetaPhor agarose
- 10 g regular agarose
- 50 ml 10X TAE buffer
- ddH2O to 500 ml. Mark a line on the flask at the 500 ml volume.

2. Autoclave the flask for 20-30 minutes. 

3. Pour the solution into autoclaved flasks or bottles in 100 ml volumes when the solution cools slightly. If the volume is below the marked 500 ml line, add additional TAE buffer before pouring.

4. To cast the gel, gradually microwave a flask or bottle with 100 ml of the agarose mix. Heat the mix until it is melted but cool enough to pour.

5. Pour the mix into the gel molds. It takes approximately 40-45 ml for one gel. 

6. Prepare a normal 3% agarose gel as well with 1.5 g agarose in 50 ml TAE buffer.

7. Prepare the 20 ul samples with 4 ul 6X loading dye.

8. Add 12 ul of prepared sample to each gel as indicated in table 2.

9. Run the gel until the bands have migrated at least ⅔ of the gel length. 

10. Stain the gel with EtBr and visualize it with UV.



Cas9 Condition Optimization

The usefulness of PCR extends beyond replicating a target segment. Unfortunately, PCR cloning can be difficult due to the 3’ adenosine bases added to the ends of PCR products by Taq polymerase. This experiment details cloning a gene from S. cerevisiae such that it is compatible with the TOPO TA cloning process. These adenosine overhangs can be used in TOPO TA cloning to easily ligate the PCR products into a specialized vector that is ready for transformation in competent cells. 

PCR
1. Label 2 PCR tubes as 50 ng and 100 ng yeast genome DNA.

2. Label 2 PCR tubes as no DNA control and no primer control.

3. Add 2 ul primer and 2ul dNTP to each tube.

4. Add 50 or 100 ng of the appropriate plasmid to the appropriate tubes.

5. For each tube, add 5 ul 10X reaction buffer and 0.25 ul of Hotstart Taq DNA Polymerase, and ddH2O to bring the total volume to 50 ul. 

6. Place PCR tubes in thermocycler and use the following settings:

- 94 C for 1 minute

- 94 C for 1 minute, 62 oC for 1 minute, 72 C for 2 minutes (30 cycles)

- 72 C for 5 minutes

- Hold at 10 C


TOPO Cloning
1. Combine the cloning reagents as shown in table 1. Ideally include one reaction with only the pCR 2.1 vector as a control.

TOPO TA Cloning

This protocol details a typical setup for screening for transformed cells in the process of cloning a gene following a typical cloning procedure. An alkaline phosphotase is use to ensure there is no self ligation of the vector. 

α-Complementation - Many shuttle vectors contain an ampicillin resistance gene so only successfully transformed cells may grow on ampicillin containing media. They also contain the β-galactosidase α-fragment. Using functional complementation, the expression of this fragment in cells with the lacZΔM15 mutation allows functional expression of β-galactosidase. Cells expressing this enzyme can hydrolyze the modified sugar X-gal into a blue product, so transformed colonies can be easily identified by their blue color. As the β-galactosidase α-fragment typically lies within the MCS of the lacZ gene, successful DNA insertions into the vector plasmid stops functional expression of the β-galactosidase gene. Thus cells transformed by plasmids containing an MCS insert appear white and uncolored. This protocol also details the use of IPTG, a non-metabolizable galactose analog, to induce expression of the lacZ operon. Both IPTG and X-Gal will be incubated with the LB-Amp plates before inoculation with transformed bacteria.

Alkaline Phosphatase Treatment

1. Add 1 µl of enzyme and 1.5 µl digested vector to a microfuge tube.

2. Add ddH2O to bring the total volume to 20 µl. 

3. For a control untreated vector, add 1.5 µl digested vector and enough ddH2O to bring the volume to 20 µl to a microfuge tube.

4. Incubate the reactions for 1 hour at 37 oC

5. For both the treated and untreated vector, add 100 µl of DNA binding buffer to the 20 µl samples and vortex.

6. Transfer solution to Zymo-Spin column placed in a collection tube. Any miniprep spin column will as long as it is usable for the length of dsDNA you are treating.

7. Centrifuge column at ≥10,000g for 30 seconds and discard flow through. 

8. Add 200 µl Wash buffer and centrifuge at ≥10,000g for 30 seconds. Repeat for a second wash.

9. Incubate column with 50 µl elution buffer for 1 minute. Move column to a microfuge tube and centrifuge at ≥10,000g for 30 seconds.

10. Reapply eluate to Spin column and incubate for 1 minute. Spin for 30 seconds at ≥10,000g to elute final product. 

Ligation of Insert

1. Add 50 ng of the vector (treated and untreated) and a 3-fold molar excess of the insert to a microfuge tube.

2. Add 50 ng treated vector without insert to a microfuge tube for the vector only control.

3. Add dd H2O to each tube to bring the total volume to 10 µl. 

4. Add 10 µl 2X NEB Quick Ligation Buffer to each tube.

5. Add 1 µl NEB Quick ligase to each tube. Mix the reaction by pipetting up and down.

6. Incubate at room temperature for 5 minutes.

7. Move the reaction tubes to an ice bath. 

Transformation of E. coli

1. Prepare the water bath at 42 oC and the agar plates at 37 oC.

2. Thaw 3 tubes with competent cells in ice bucket. 

3. Add 2 µl of each different ligation reaction to each tube. Be sure that the added volume is less than 5 µl. Tap the tubes gently to mix.

4. Incubate the tubes for 30 minutes on ice.

5. Heat shock the tubes in the water bath for 30 seconds.

6. Place the tubes back on ice for 2 minutes.

7. Add 250 µl SOC media to the tubes using aseptic techniques. Incubate the tubes at 37 oC and 225 – 250 rpm on a shaker for 1 hour. 

8. media. Place on the shaker with the sample tubes and incubate for 1 hour.

9. Add 240 µl IPTG stock and 240 µl X-Gal stock to a microfuge tube. Mix gently.

10. Add 80 µl IPTG X-Gal mix to 6 LB-Amp plates. Spread the mixes over each plate using aseptic technique and a glass spreader. Incubate the plates at 37 oC for 20 minutes.

11. Plate 50 µl and 150 µl from each tube onto two separate LB-amp plates, for a total of 6 plates. 

12. Incubate the plates overnight at 37 oC.

 
The AP treated vectors cannot form a circular plasmid without the presence of an insert, so your treated vector + insert reaction should contain only recombinant circular vector. Bacteria transformed with this reaction mix have a non-functional β-galactosidase, and thus appear white. If any bacteria happen to have innate ampicillin resistance, white colonies will appear on plates streaked with these cells. Untreated vector in still has 5’ phosphates, so the reaction creates both nonrecombinant plamids that recircularized without an insert, as well as recombinant plasmids with the insert. The nonrecombinant plasmids create blue colonies due to alpha complementation restoring β-galactosidase function, while the recombinant plasmids create white colonies due to the disruption of the β-galactosidase gene. 



X-Gal Blue White Screening

PCR can be performed directly from a scraped cell colony, using an extended denaturing step to cause lysis of the cells. This is a nice time saving protocol for PCRing targets directly from antibiotic selective plate media. 

Colony PCR with Patch Plating
1. If subculturing via patch plate, divide a warm LB-Amp plate into a grid (commonly 3x3), labelling each square on the back of the plate.

2. Using a sterile wooden stick, aseptically pick up a transformed colony and smear some of the colony in a PCR tube. 

3. If subculturing via patch plate, use the same stick to patch it on one of the squares. 

4. Repeat steps 2 and 3 for multiple colonies.

5. Place patch plate in 37oC incubator. Move to a 4oC fridge the next day.

6. Add reagents to the PCR tubes as described in table 1. The reagent volumes can be modified to fit whatever your typical PCR setup looks like.

7. Run the PCR cycles as detailed in table 2.

8. The resultant PCR products can be run through a gel for gel extraction or cleaned with a PCR cleanup kit.

Figure 1 displays the results of the colony PCR using all 6 patched colonies. Lanes 1 - 6 contain the colony PCR products of 6 transformed colonies. A 1 kb ladder was loaded into lane 7. Lanes 8 and 9 were PCR products of untransformed control colonies. 


Colony PCR

Gel extractions can be a low yield affair, but overnight incubation over a spin column can increase yields. This protocol details a modified QIAquick Gel Extraction protocol.

QIAquick Gel Extraction Kit 

1. Cut the desired band out of an agarose gel using a scalpel. 

2. Weigh the slice in a snap cap tube and add 3 volumes of QG buffer per volume of gel, assuming the gel density is roughly 1µl/mg. This can be whatever gel dissolving buffer (with chaotropic salts and guanidine thiocyanate) you typically use.

3. Add 1 volume of isopropanol to the tube and vortex.

4. Place a QIAquick spin column in a collection tube. Add the sample to the column and incubate over column overnight at room temp. 

5. Centrifuge spin column for 1 minute at 13,000 RPM. 

6. Discard flow through. Repeat step 5 if the sample volume was greater than 800 µl.

7. Add 750 µl of buffer PE to column and centrifuge for 1 minute at 13,000 RPM. Discard flow through.

8. Centrifuge for an additional 1 minute to remove all wash buffer.

9. Place column in collection tube and add 50 µl EB buffer or water directly to the silica portion of the column. Incubate up to 4 minutes then centrifuge the column for 1 minute to elute DNA.

Analyze DNA using a standard Nanodrop/general spectrophotometer protocol for 260/280 absorbance ratios. Store all samples in a -20 oC freezer.

Higher Yield Gel Extractions

Since working in a genomics lab, one of the nifty tricks I learned was how to re-use the Agilent Bioanalyzer 2100 DNA chips (Cat No.5067-4626) The chips are the limiting reagent and you can get quite a bit of use out of your gel by doing this method. It also means, in a pinch, if you are running out of chips or reagents that you can always have some around to run the bioanalyzer. It’s a pretty simple process but has saved me a bunch of times (and countless dollars). I’ve used it for running gDNA (diluted of course), and libraries.

You can follow the protocol from this paper that outlines it and download it from their supplementary materials (https://www.future-science.com/doi/full/10.2144/000114232). The protocol is perfect and I’ve run it as is with no problems. Some of the chips I’ve even used up to 20 times without fail. I’ve tried it with RNA chips before too but it’s quite finicky what with the easily degradable nature of RNA. I never did protein work so not sure how it works in that realm.


Molecular Biology

Disciplines/Biology

Community Guidelines

Have a question?