Fall petB intron team

all present!

Fall atpF intron team

not pictured: Alivia McMahon

Purifying samples for sequencing

Research Protocol:  Direct Purification of DNA from PCR Amplifications Using the Wizard PCR Preps DNA Purification System (Promega Corp.) with a Vacuum Manifold

 

If your chloroplast region-PCR reactions worked (single band of sufficient size and yield), you can proceed to the next procedure of purifying them in preparation for DNA sequencing.  Some form of PCR purification is recommended prior to DNA sequencing.  DNA sequencing reactions include materials that are very similar to those included in PCR and sequencing requires that these components be in appropriate concentrations.  Significant carryover of PCR components disrupts this balance and can cause poor sequencing reaction results.

If you saw small products (< 50 bp) near the bottom of your PCR sample gel run, these are most likely primer artifacts which are generated from the primers interacting with each other or with themselves (aka primer dimers).  The following purification procedure not only removes any excess PCR components it also produces very poor yields of products < 75 bp (3% recovery).  If performed correctly, products in the expected size range of our targets (around 1000 bp depending on region and species), the expected recovery is approximately 96%.

If other products are co-amplified with your products that would not be removed by this procedure AND are the result of non-specific primer binding, then you can simply use primers that will bind to sites within the amplified sequence.  This should allow sequencing of just the desired product.  However, if you are dealing with a case of heteroplasmy, multiple alleles or multiple loci (common for nuclear genes), cloning the PCR products into a plasmid might be necessary before DNA sequencing.  If these products are of a different size (can be separated on a gel), this kit can be used for gel purification – the PCR products are isolated from excised pieces of the agarose gel).

As our samples tend to amplify with high yield and little background, we cleaning them directly and will be using the same primers for sequencing as were used for PCR.

Before beginning the following procedure, first verify which of your reactions was successful.  If both amplifications of a DNA sample worked, you will combine them as part of the purification procedure.

 

Put on gloves!

 Step 1: For each successful amplification from a sampled species, please label one 1.5 ml tubes with the DNA code and region. You can use from 30-300 ul of PCR product in one purification.  For specific DNAs where both amplifications were successful, add both to this tube. Since we performed 100 ul reactions and used 5 ul for verification, 95 ul remains (2 x 95 ul = 190 ul).

Step 2: Add 100µL of Direct Purification Buffer.  Vortex briefly.

Step 3: Add 1ml of resin (make sure resin is suspended in the stock bottle PRIOR to pipetting.  [Warning:  wear gloves!  The resin mix contains Guanidium thiocyanate, a powerful protein denaturant.]

Step 4: Close the tubes and vortex 3 times over the course of 1 minute (about every 20 seconds).

Step 5: For each sample tube, obtain a Minicolumn and Syringe Barrel.  Attach a minicolumn and syringe together via the Luer-Lok (they screw together). [Label these the same as the tube!!]  Take each of these to the vacuum manifold (blue “pig”) and insert the Minicolumn end into one of the connections on top of the manifold.

Step 6: Pipet the resin/ DNAs into the appropriate syringe barrel.  Turn on vacuum and let the resin be drawn into the minicolumn (to the point that the liquid is removed).  If some are drawn through faster than others, turn the top-cock to stop the pull of the vacuum; when all are completed, turn off vacuum. [Under the chemical conditions of the buffer/resin, DNA binds to the resin.  The % recovery depends on how well it binds or remains bound during the next step.]

Step 7: To wash the column, add 2ml (2 x 1000 ul) of 80% isopropanol then reactivate vacuum and wait until the isopropanol has exited the Minicolumn.  Again, shut stop-cocks as necessary. [This step removes salts and other materials non-specifically bound to the resin.]

Step 8: Dry resin by activating the vacuum for another 30 seconds.  Then, remove and transfer Minicolumn to a fresh 1.5ml tube. [Drying the resin is important as residual isopropanol will inhibit DNA sequencing.  However, over-drying could lower recovery rates.]

Step 9: Centrifuge Minicolumn at 10,000 rcf (x g) for two minutes. [This step ensures that all isopranol is removed – but does not risk over-drying.]

Step 10: Transfer Minicolumn to a new 1.5ml tube and pipet 50µL of H₂O (filter sterilized deionized) and wait a minute. After the waiting is over, centrifuge for 20 seconds at 10,000 rcf.

Step 11: Discard Minicolumn, close tube.

Step 12: Quantify using NanoDrop Spectrophotometer (1 ul).  Determine three team members who can 1) collect  quantification data (DNA code and quantity of purified) from all team members, 2) organize into a clear table and 3) post this table to the course blog (under the appropriate chloroplast region PCR category).  The role of each of these three team members (and the help of any other member of the team) should be noted at the bottom of the blog post.

 

The desired yield (proposed by the vendor who performs the sequencing) is 10 ul of sample at 50 ng/ul.  However, if your yield is less than this, sequence can still be obtained.

Store all DNAs at -20°C (freezer).

DNA purification and pre-sequence quantification.

Prior to purification, successful PCR replicates were combined and each pair of students was given 3-4 of these samples to purify using the Wizard PCR Purification system (Promega).  After completion of the protocol, 1 ul of each sample was quantified using a Nanodrop Spectrophotometer.  The results are shown in the following tables (italicized concentration values indicate samples considered too dilute to submit for sequencing).  Purified samples and the same primers used for PCR were submitted for sequencing to Functional Biosciences.  The facility requests that the purified PCR products shipped to them be at least 50 ng/ul.  However, they will make adjustments for samples that are more or less concentrated.

Sample ID	ng/ul	A260	A280	260/280	260/230
3-1 atpF	60.5	1.21	0.77	1.56	0.22
3-2 atpF	59.01	1.18	0.6	1.98	0.12
3-3 atpF	7.3	0.15	0.1	1.53	0.04
3-4 atpF	47.65	0.95	0.5	1.92	0.08
3-5 atpF	61.47	1.23	0.64	1.92	0.13
3-6 atpF	16.28	0.33	0.29	1.13	0.04
3-7 atpF	91.7	1.83	0.96	1.9	0.16
3-8 atpF	76.36	1.53	0.79	1.93	0.12
3-9 atpF	74.24	1.49	0.89	1.66	0.24
3-10 atpF	84.42	1.69	0.9	1.87	0.26
272 atpF	138.4	2.77	1.69	1.64	0.34
278 atpF	84.82	1.7	1.18	1.43	0.28
300 atpF	5.05	0.1	0.08	1.33	0.02
366 atpF	81.16	1.62	0.86	1.89	0.14
382 atpF	68.63	1.37	0.7	1.95	0.11
384 atpF	9.39	0.19	0.08	2.36	0.02
387 atpF	76.53	1.53	0.79	1.95	0.13
389 atpF	50.61	1.01	0.52	1.94	0.09
Sample ID	ng/ul	A260	A280	260/280	260/230
3-1 petB	22.2	0.44	0.21	2.15	0.05
3-2 petB	85.42	1.71	0.87	1.96	0.14
3-3petB	18.7	0.37	0.21	1.83	0.05
3-4 petb	78.95	1.58	0.82	1.93	0.15
3-5 petB	242.8	4.86	2.94	1.65	0.55
3-6 petB	20.36	0.41	0.22	1.85	0.07
3-7 petb	162.4	3.25	1.65	1.96	0.3
3-8 petB	187.1	3.74	2.1	1.78	0.36
3-9 petB	40.5	0.81	0.4	2.02	0.06
3-10 petB	69.34	1.39	0.71	1.94	0.11
272 petB	141.3	2.83	1.76	1.6	0.38
278 petB	71.9	1.44	0.74	1.95	0.12
300 petB	41.56	0.83	0.42	1.99	0.08
364 petB	79.06	1.58	0.81	1.95	0.15
366 petB	102.8	2.06	1.09	1.89	0.24
376 petB	74.36	1.49	0.79	1.89	0.13
382 petB	91.59	1.83	0.94	1.95	0.16
384 petB	30.11	0.6	0.31	1.97	0.05
387 petB	150.1	3	1.67	1.8	0.26
389 petB	119.3	2.39	1.5	1.59	0.36

New Extractions for Fall 2011

Twelve species representing the various subtribes of the orchid tribe Vandeae were extracted on Sept. 13, 2011.  These DNAs would eventually be used as templates from which to amplify the introns of the atpF and petB genes of the plastid genome.  These DNAs were extracted, electrophoresed and quantified as described in the Techniques portion of this blog.

While it is a sign of a high quality extraction (good technique resulting in limited fragmentation of the DNA), this is not a strict requirement for achieving success in PCR (at least with amplicons of a comparatively small size).  High quality extraction is typified by most of the DNA being in a large band with minimal to no smear of smaller fragments.  Causes of smearing could be excessive tissue maceration while grinding or allowing the sample to thaw in the mortar prior to transfer to microcentrifuge tubes and addition of the lysis buffer.  The latter error could allow DNases to be active.

Variation in yield could have a variety of causes:  incomplete transfer of ground tissue from mortar to microcentrifuge tube, excessive degradation (DNases), or high levels of polyphenolics or polysaccharides.  While the extraction kit used claims to address the latter issues, excessive amounts could still overwhelm the system.  However, low yielding extractions can still provide sufficient DNA for amplification of plastid targets which exist in high copy numbers per cell.

 

1% agarose gel with DNAs 3-1 to 3-6

Lane	ID	Species or Marker	ng/ul
1		Lambda-HindIII
2	3-1	Phalaenopsis tetraspis	8.75
3	3-2	Sedirea japonica	16.84
4	3-3	Cleisostoma linearilabatum	4.17
5	3-4	Plectrelminthus caudatus	14.31
6	3-5	Micropera pallida	12.83
7	3-6	Schoenorchis juncifolia	13.35

1% agarose gel with DNAs 3-7 to 3-12

Lane	ID	Species or Marker	ng/ul
1		Lambda HindIII
2	3-7	Holcoglossum wangii	5.43
3	3-8	Polystachya zambesiaca	3.14
4	3-9	Pomatocalpa bicolor	13.42
5	3-10	Podangis dactyloceras	5.66
6	3-11	Cleisostoma crochettii	17.61
7	3-12	Ceratochilus biglandulosus	7.76

 
Aliquots of each DNA were diluted in DNA Dilution buffer (10 mM Tris, 0.1 mM EDTA, pH8)to 1 ng/ul for subsequent PCR amplification.

Ceratochilus biglandulosus

Ceratochilus biglandulosus   Blume is a small member of the subtribe Aeridinae found growing on mossy trees at high elevation in Java and Sumatra. Ceratochilus is one of several monotypic genera in the Aeridinae.  This means that it currently only contains one species.   The succulent leaves are roughly 1.5 – 2 cm in length while the bright white, crystalline flowers, produced one per inflorescence, are about 3.75 cm wide.

Small orchid species with comparatively large showy flowers are popular with connossieur orchid collectors and, as such, C. biglandulosus is not difficult to obtain from US orchid vendors.   However, it can sometimes be a challenge to maintain as it does not tolerate long periods of dryness or to be overly wet.   Therefore, regular watering and a well draining media are optimal.  This species prefers bright shade and intermediate to warm temperatures.

Using both plastid and nuclear sequences, Hidayat and co-workers (2005) suggested that Ceratochilus was a member of the “Trichoglottis alliance” along with the genera Staurochilus, Trichoglottis, Vandopsis, and Ventricularia.

specimen used for DNA extraction

http://galerie.orchideen-forum.de/albums/userpics/10003/ceratochilus_biglandulosus.jpg

 

 

 

 

 

 

 

 

 

http://orchids.wikia.com/wiki/Ceratochilus_biglandulosus

http://www.orchidspecies.com/ceratbiglandulous.htm

Hidayat, T., T. Yukawa and M. Ito. 2005.  Molecular phylogenetics of subtribe Aeridinae (Orchidaceae): insights from plastid matK and nuclear ribosomal ITS sequences.  Journal of Plant Research 118: 271-284.

Agarose Gel Electrophoresis

If you are looking at this post, you probably need to actually pour, load and run an agarose gel.  First off, agarose is one of the polysaccharides found in agar, a seaweed product that, when melted, has the consistency of stiff Jello.  In fact, not only is agar an ingredient in semisolid culture media for microbes,  it is also a vegetarian alternative to gelatin.

Agarose gel electrophoresis is a simple method for verifying the status and getting a qualitative estimate of the yield of extracted nucleic acids (DNA or RNA).  It can also be used for separating nucleic acids of various sizes, such as DNA following digestion with restriction enzymes.  It can also be the first step in a more involved procedure such as Southern or Northern blot analysis in which the nucleic acids are transferred to a supportive membrane and then hybridized with probes to identify specific fragments (Southern) or specific transcripts (Northern).  We are going to use this method to check our extracted DNAs and to determine the success of our attempts at PCR.

Below you’ll find a description of the specific buffer system and procedure used to prepare and pour a gel.

BUFFERS (Gel and Running)

We’ll be using a “FastGel” buffer system promoted by Promega, Inc.  It allows gel with a single comb to run to completion in about 20 min.  This is because the buffer combination enables the use of higher voltages (200 V)  for a short period of time.  This voltage with the standard 1x/1x buffer system would quickly lead to excessive heat and melting of the gel.

If you need to run a gel and the working stocks are out or low, the following recipes will allow you to make more.

1X TAE (gel buffer)

In the fridge are several small bottles with 50X TAE.  These are stocks that you will use to make either the gel or running buffer.  To make the 1 L of 1X gel buffer, measure 20 ml of 50X TAE and add it to 980 ml of deionized water (in the large container near one of the lab sinks).  Mix well before using.

0.25 X TAE (running buffer)

To make 500 ml of running buffer, add 2.5 ml of 50X TAE to 498 ml (close enough) of deionized water and mix well.  Or, you can add 250 ml of well-mixed 1x TAE to 750 ml of dei water.

THE GEL

Pouring a 1% agarose/1x TAE gel:

1. Obtain a 250 ml Erlenmeyer flask
2. Measure out 1 g agarose and 100 ml of 1X TAE (gel buffer)
3. Pour the agarose and approximately 80 ml of the gel buffer into the flask and swirl
4. Allow the agarose to hydrate for about 5 min. (this allows for more even melting)
5. Place a small Erlenmeyer into that holding the agarose/buffer mix (rattling of the small flask will alert you that the solution is boiling)
6. Microwave the solution at roughly 30 sec intervals until the agarose is completely melted (hold the flask up into the light to verify that all agarose beads have melted).  Use several layers of paper toweling to protect your hand from the heat and be careful when you first grip the flask in case the hot agarose boils over/sprays!
7. Add the remaining 1x TAE to the flask and swirl (this will help the mix cool down faster).
8. Checking every few minutes, let the solution cool until you can stand to hold onto the flask without burning yourself.  At this point, it is cool enough to pour into the gel molds (see the next section for help on setting up the gel mold) without either damaging the mold or burning you.
9. Let the gel cure for at least 20 min.
10. If you are doing this a day early, wrap the gel in the mold in plastic wrap and place in the fridge until you are ready to use it (if the lab supply of combs is running low, remove the comb(s) for other to use).

Setting up a gel mold:

1. Obtain a gel mold and make sure that the end walls are positioned so as to create a four-sided box in which to pour the agarose (make sure the screws are secure – do not tighten too much, the screws are plastic and can break!).  If you are concerned that the mold still might leak, you can carefully add lab tape  around each end of the mold so that the end-wall/mold body is sealed.
2. If you are just running your set of reaction, place a comb (10 or 12 well) in the upper comb slot.  If you and another group wish, you can add a second comb. [you cannot insert combs once the gel has set!]
3. Place the mold with combs on a level surface and pour in your warm agarose mix.

When the gel is ready to load

1. Once the gel is cured, it will have become opaque (cloudy milky in color) and be fairly stiff to the touch.  At this point you can carefully remove the combs.
2. Carefully remove the combs by pulling them slowly straight up.
3. Loosen the screws that hold the end walls in place (Do NOT remove them!).
4. Push then end walls down so that the agarose at the top and bottom is now exposed.  (You can re-tighten the screws to help keep them in the lowered position.)
5. Place the gel on the platform in the gel rig. [Make sure that the wells are closest to the negative (black) electrode so that the DNA can migrate towards the positive (red) electrode.  Unless the gel rigs have been moved, this normally means the wells in the top half of the gel are farther away from you than the other end of the gel.
6. If needed, add enough 0.25 X running buffer to just cover the gel AND to fill the wells.

Orchid DNA Extraction Protocol

Step 1: Orchid Total DNA Extraction (DNeasy Plant Mini Kit – Qiagen)

1.      Set volumes on two pipettors: 400 ul and 4 ul.  Obtain gloves.

2.      A. Record species name (check spelling!)

B. Obtain and label a microcentrifuge tube.

C. Carefully remove a small amount (< 100 mg (0.1 g)) of tissue (leaf tip on a younger leaf) using a fresh razor blade.

D. Finely dice tissue.

E. Obtain mortar/pestle from freezer, place tissue in mortar, carefully cover with liquid nitrogen.

F. Start by slowly crushing tissue until liquid nitrogen has almost evaporated.

G. Crush tissue by rotating pestle… grind tissue into a uniform fine powder.

H. Using spatula, scrape powdered tissue into tube as quickly as possible.

 Note: Work quickly to avoid thawing of tissue.  There is less chance of DNase damage if tissue remains frozen.

3.      A. Add 400 ul of AP1 and 4 ul of RNase A to sample.

B. Vortex to mix (no tissue clumps should remain) and incubate at 65 C for 10 min.

C. Invert 2-3 X during incubation (IMPORTANT!)

This step lyses the cells and removes RNA.

4.      Add 130 ul of AP2, mix and incubate on ice for 5 min.

This step precipitates detergent, proteins and polysaccharides.

5.      Centrifuge to pellet all insoluble materials at 20,000 x g for 5 min.

 This step minimizes DNA shearing that can result if lysate is viscous (and passes through spin column – next step).  DNA is in the supernatant.

6.      A. Transfer supernatant into QiaShredder Mini spin column in a 2 ml collection tube.

[Keep carryover of pellet material to a minimum.]

B. Spin at 20,000 x g for 2 min.

7.      Transfer the flow-through into a new tube (set pipettor to 450 ul).  If you have significantly more or less supernatant, adjust pipettor until you can get a more accurate estimate of flow through volume.

8.      Add 1.5 volumes of AP3 and mix well by inversion.

9.      A. Place a DNeasy Mini column into a 2 ml collection tube.

B. Pipet 650 ul of supernatant-AP3 mixture into Mini column

C. Spin at 6000 x g for 1 min.

D. Discard flow-through

          E. Repeat 9B-D with remaining mixture and discard the collection tube (NOT the column)

This step facilitates binding of the DNA to the DNeasy minicolumn filter

10.    A. Place the DNeasy minicolumn in a new 2 ml collection tube.

B. Add 500 ul Buffer AW

C. Spin for at 6000 x g for 1 min.

D. Discard flow-through (keep the collection tube!)

11.    A. Add 500 ul Buffer AW to the mini column

B. Spin at 20000 x g for 2 min.

 These steps (#10 and 11) “wash” the DNA to remove salts.  The higher speed spin assures removal of residual alcohol from DNA and dries it.

12.   A. Remove the minicolumn to a 1.5 ml centrifuge tube. (avoid any carryover of flow-through!)

B. Add 100 ul of Buffer AE onto the minicolumn membrane (Do NOT touch membrane with pipet tip!)

C. Incubate at room temperature for 5 min.

D. Spin at 6000 x g for 1 min.

13.    Repeat 12B-D and discard minicolumn

 These steps (#12 and 13) elute the DNA from the column.  The DNA is now highly purified.  We will check quality  via gel electrophoresis and quantify the DNA using a NanoDrop Spectrophotometer.  If sufficient yield and purity, it can be used for a variety of downstream applications. 

 Step 2:  Use 5 ul of extraction for gel electrophoresis.  Only 1 ul is required for quantification (use Buffer AE as the blank!!).

 DNA largely in the form of high molecular weight fragments is most desirable.  This will appear as a bright large band with a minimal “smear” of smaller fragments.  While PCR is very robust even when DNA is not in this optimal state, high molecular weight DNA is indicative of good extraction technique (and an extraction kit that is working well).

 With this kit, yield of 6 – over 20 ng/ul are typical.  I have found that 10 ng of total DNA is sufficient for amplification of plastid targets. To simplify set up of PCR reactions, aliquots of extracted DNAs will be diluted to 1 ng/ul.  10 ul of these dilutions is then used in each PCR reaction.

Introduction