Author Archives: Allan

Sunflower seed sterilization

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Hi all,

Here is a seed sterilization protocol that surprised me – No fungus visible after 4 -5 days of growth on nutritious media.

In 100 ml distilled water mix the following

1 g sparkleen soap powder (dish washing stuff) –> This is the top end for sparkleen.  You may need to use less.
2 mL bleach (final concentration = 2%)
2 ml PPM. (final concentration = 2%)

Sterilize in 15 ml tubes.

Rinse seeds  with autoclaved water 3x.

Results: Normally the first image would contain many fungal blooms.  Not so in the images below.

Friday November 21st

WP_20131122_15_51_06_Pro

 

Monday November 26thWP_20131125_001

Plant DNAzol

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Hi all,

Here’s another method for DNA extraction.  The blog is stuffed to the gills with DNA extraction methods.  The current standards are ‘Qiagen-like’ columnless – used to generate the DNA for the genome sequencing project and CTAB.  I add this protocol because itis
easy and it is effective.

I extracted from Ha89 and harvested ~ 115 ng/uL from 20 cm tall plants.  I also purposely ‘took it slow,’ letting tissue thaw after freezing to see PlantDNAzol’s efficacy.  It’s efficient.

PlantDNAzol-uncut

Quality is good

The gel to the left:

Right most lane contains Ha89 genomic DNA – 10 uL loaded of 70 at 115 ng/uL DNA – 260/280 was 1.78.  260/230 was ~1.00 (I suspect I could have added an additional ethanol wash to remove Guandine from the DNA mixture.

Is the DNA useful?  Can downstream reactions proceed?

Yes.  I digested the DNA with a methylation sensitive restriction enzyme, PstI and a methylation insensitive enzyme, EcoRV

PlantDNAzolcut-22hrs

<–The gel to the left :

Leftmost lane  Ladder

A vector digested with PstI,

Ha89 gDNA digested with PstI 240 minutes,

Ha89 gDNA digested with EcoRV 240 minutes.

The DNA digests.  But does it contain contaminants that upset the enzymes over long incubations?  Overnight?

PlantDNAzolcut-240minutes

Gel above:  From left- Ladder, unrelated vector digest,

Ha89 gDNA digested with PstI 22 hours

Ha89 gDNA digested with EcoRV 22 hours.

Protocol for DNAzol extraction (exactly as published by LifeTech but easier to follow – http://tools.lifetechnologies.com/content/sfs/manuals/10978.pdf:

Have these items on hand:

1 0.6 mL DNAzol per 100 mg sample

2. 0.3 mL chloroform per 100 mg sample

3. Timer

4. 100% ethanol (0.225 mL per sample),

5. 75% ethanol (0.3 mL per sample)

Handle all inversions carefully.  When you see invert or shake handle your samples gently

1. Mix 100 mg ground tissue with 0.3 mL PlantDNAzol – 100 mg is max.  Overdoing will hurt your yield.

2. Invert gently to aid in lysis and dispersion

3. Once completely dispersed incubate at RT, 5 min, shake periodically.

4. Add 0.3 mL chloroform and mix.

5. Once completely dispersed incubate at RT, 5 min, shake periodically.

6. Centrifuge at RT, 12 000 g (NOT rpm) 10 min

7. Harvest the supernatant. – you’ll see a phenol/chloroform styled triple layer.  The middle layer will be pulpy containing your cellulosic debris and proteins.  Don’t collect the middle layer.  Less is more

8. Mix supernatant from 7 with 225 uL 100% ETOH.

9. Incubate at RT, 5 min

10. Centrifuge mixture 5000 g 4 min – get preparing for step 11

11. Make a PlantDNAzol – Ethanol mixture:  For one sample mix 0.3 mL PlantDNAzol with 0.225 mL 100% ethanol.

12. Discard the supernatant from 10 and mix it with 0.3 mL of the mixture prepared in step number 11

13. Incubate as in step 9.

14. Spin as in step 10.

15. Pour off supernatant

16 Wash pellet with 75% ethanol – 0.3 mL – this step can be repeated if your 260/230 isn’t adequate.  Guanidine absorbs strongly in 230 nm wavelength

EDIT: repeat step 16 for a total of 2 washes.

17. Spin as in step 10.

18. Remove supernatant – if your samples are green repeat step 16.

19. Resolubilize your DNA in TE or NaOH.  Make sure to run your bead of TE over the wall of the tube to collect your DNA.

 

EDIT – Less is more as is usually the case with DNA extraction.  I harvested from 38, 60, and 100 mg of tissue.  – A sweet spot for tissue quantity is 45 to 60 mg for the given amount of Plant DNAzol

Qubit quantifiication:  45 mg of tissue yielded 264 ng/uL ug/mL.  60 mg, 305 ng/uL.  100 mg, 12.4 ng/uL

PstI digest tests

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Edit: I forgot to thank DanB and Kate for the generous donation of DNA.  Sorry guys 🙁

Edit 2: After the lab meeting some questions have been asked.

To summarize :

1.  All the enzymes work equally well.  There is a slight performance decrease if one uses the PstI-HF.  

2.  I’d recommend using the PstI from Invitrogen – Invitrogen works extremely well and is the cheapest among the Psts tested ( $22 versus $75  ).  EDIT: Brook and Kate let me know that I failed to factor in the units provided for a given dollar value.  Invitrogen provides the cheapest enzyme.

For NEB – With our volume purchases we get 10000U for $71.40.

For Invitrogen – With our volume purchases we get 10000U for $63.90 {represents price negotiated with Helen, accounts manager at Invitrogen Stores}

Thanks guys for pointing that out!

This adds up considering how much we’re going through.

3. Sunflower gDNA does not digest completely in 3 hours.  It is recommended to go overnight or 18 hours with your digestion.

4. After 18 hours do not be alarmed by incomplete digestion.  This is OK according to RFLP work performed by Loren.  There should be a sufficient number of fragments for GBS libraries.

Hi all,

Here are the results from the PstI digest tests:

All enzymes tested fail to fully digest sunflower gDNA.

Enzymes from Invitrogen, Thermofisher/fermentas, NEB (non HF) and NEB HF were tested.  All performed equivalently.  I would say the NEB HF was slightly less processive after 70 min.  See attached PDF for gels and full documentation of reaction conditions.

**** 2013-Oct-08-debono-digesttest *****

Thanks

Allan

NEB HF enzymes – regarding library preps

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Hi guys,

Just a note about PstI-HF:

The PstI-HF sold from NEB is less processive than the old NEB PstI/red stripe version (go here to see it: https://www.neb.com/products/r0140-psti) .  When digesting ultrapure, high concentration homogeneous DNA (plasmids) digestions fail to go to completion even when left overnight (> 16 hours).  What does this mean for you?  It means that your genomes which are more difficult substrates to deal with will not digest fully unless left to go overnight.  If you want to ensure a good digest use a positive control and switch to the linked product.

 

 

 

 

Ph*cking phenolics!

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Hi guys,

I have extracted sunflower DNA a dozen times now.  I get one consistent problem a gelatinous film coprecipitating with my DNA (carbohydrates/polysaccharaides).  I also get sharp smelling phenolic compounds.  These occur regardless of DTT/2-BME addition.

I found a paper detailing the use of 2-butoxyethanol (2-BE) to remove strawberry polysaccharides and phenolics.  The researcher’s idea is to perform a two-step precipitation:

Continue reading →

Chemical safety: a quick read

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Chemical safety: a quick read

Hi everyone,

Here’s a sobering blog post I saw at BiteSizeBio.  I use most of these chemicals regularly. Scarier still I had no idea chloroform becomes phosgene once metabolized.  The list contains popular chemicals in the Rieseberg lab.

original link: http://bitesizebio.com/articles/ten-bad-chemicals-in-the-lab-and-what-they-do-to-you/

Ten Bad Chemicals In The Lab and What They Do To You!

Continue reading →

Freeze and squeeze DNA extraction from gel

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Hi all,

Here’s a really easy DNA gel extraction:

1. Go about cutting out your bands of interest in the usual way.

2. Next cut the gel band(s) into tiny cubes.

3.  Place tiny cubes in an eppie.

4. Put eppie in -20oC for 15 to 30 min.  The idea is to make the band freeze into a mini ice cube.

5.  Knock out all of the gel band/cubes onto a 10 (long) x 5 (wide) cm piece of parafilm.

6. Fold the parafilm in half along the length while keeping your gel cubes along the fold in the parafilm.  You should have a piece of parafilm folded lengthwise with gel cubes sandwiched between two layers of plastic.

7.  Crush/squeeze the liquid out of the tiny cubes.  Liquid containing electrophoresis buffer and DNA will separate from the agarose.

8.  Collect DNA/electrophoresis buffer mixture with pipet and clean it by ethanol precipitation.

 

EDIT:  Borate in the electrophoresis buffer is a ligase inhibitor.  Make sure to thoroughly wash your DNA with 70% EtOH, at least 2 times.

Room Temperature RNA extraction

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Derivation: The following protocol is a modified Zhao Lai et al 2006 and TRizol Plus protocol.

Expected results: I tested the protocol with Arabidopsis seedlings and it gives quality RNA that was used for RT-PCR (non-quantitative).  I am able to achieve 75-270 ng/uL (100 uL elution – take this value with a grain of salt since it is derived from the nanodrop) of RNA from 38-48 mg of seedlings (this is approximately 20 seedlings grown for 18 days). I have yet to experience a failed RT-PCR with RNA isolated using this extraction.

Keys to success: Yield is highly linked to tissue mass and quality of tissue grind. The range of tissue mass is very small. One should use a small tissue amount and process a given sample over a column many times. Tissue is ground successfully only when it is not visible in the trizol/tissue/bead mixture. Minimize RNAse paranoia – Guanidine isothiocyanate does a good job of destroying protein structure, inactivating RNAses.

More after the jump Continue reading →

Gel doc Dropbox (Allan)

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Hi all,

I’m not sure how most of you were sharing your gels but I set up a dropbox folder within the Rieseberg gels folder.  Sending e-mails was tiresome.  If you’re interested in synchronizing your gels with your computer drag your folder(s) to the dropbox folder.  The dropbox account credentials (online and client) are as follows:

user=allandebono@allandebono.org

password=1d7hz@!

… Go through the process as usual to set up a shared folder.

Amplifying Large AT rich amplicons with Pfu type polymerases (Allan)

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This is a protocol for amplifying very large amplicons that are high in AT.  I developed in order to amplify the 8.2 Kb region containing the promoter of Arabidopsis FT which is approximately 70% AT.

In my hands it worked reliably with an 8 Kb amplicon (DeBono notebook 1, July 2010) but can be easily modified for longer products using more dNTP and optimized template concentration.

Continue reading →