Tag Archives: gel

PCR – Oly RAD-seq Prep Scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.
After determining the minimum number of PCR cycles to run to generate a visible, 166bp band on a gel yesterday, ran a full library “prep scale” PCR.

 

REAGENT SINGLE REACTION (μL) x11
Template 40 NA
ILL-HT1 (1μM) 5 55
ILL-BC# (1μM) 5 NA
NanoPure H2O 5 55
dNTPs (1mM) 20 220
ILL-LIB1 (10μM) 2 22
ILL-LIB2 (10μM) 2 22
5x Q5 Reaction Buffer 20 220
Q5 DNA Polymerase 1 11
TOTAL 100 550

 

Combined the following for PCR reactions:

  • 55μL PCR master mix
  • 40μL ligation mix
  • 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.

 

SAMPLE BARCODE SEQUENCE
Oly RAD 02  1  CGTGAT
Oly RAD 03  2  ACATCG
Oly RAD 04  3  GCCTAA
Oly RAD 06  4  TGGTCA
Oly RAD 07  5  CACTGT
Oly RAD 08  6  ATTGGC
Oly RAD 14  7  GATCTG
Oly RAD 17  8  TCAAGT
Oly RAD 23  9  CTGATC
Oly RAD 30 10 AAGCTA

 

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
17 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

 

After cycling, added 16μL of 6x loading dye to each sample.

Loaded 10μL of ladder on each of the two gels.

Results:

 

Things looked fine. Excised the bands from each sample indicated by the green arrow. Before and after gel images show regions excised. Will purify the bands and quantify library yields.

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PCR – Oly RAD-seq Test-scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.

Prior to generating full-blown libraries, we needed to run a “test-scale” PCR to identify the minimum number of cycles needed to produce the intended product size (166bp).

I ran PCR reactions on a subset (Sample #: 2, 3, 17, & 30) of the 10 samples that I performed adaptor ligations on 20151029.

PCR reactions were set up on ice in 0.5mL PCR tubes.

REAGENT SINGLE REACTION (μL) x4.4
Template 8 NA
NanoPure H2O 1 4.4
dNTPs (1mM) 4 17.6
ILL-LIB1 (10μM) 0.4 1.76
ILL-LIB2 (10μM) 0.4 1.76
ILL-HT1 (1μM) 1 4.4
ILL-BC1 (1μM) 1 4.4
5x Q5 Reaction Buffer 4 17.6
Q5 DNA Polymerase 0.2 0.88
TOTAL 20 52.8

 

Combined 12μL of master mix with 8μL of the ligation reaction from earlier today.

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
27 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

We’re following the “1/4 reduced representation” aspect of the protocol. As such, 5μL of each reaction was pulled immediately after the extension (72C – machine was paused) of cycles 12, 17, 22, & 27 in order to determine the ideal number of cycles to use. Also ran the ligation reactions (labeled “Ligations” on the gel below) of the samples as a pre-PCR comparison. Treated them the same as the PCR reactions: mixed 8μL of the ligation with 12μL of H2O, used 5μL of that mix to load on gel.

These samples were run on a 1x modified TAE 1.2% agarose gel (w/EtBr).

 

Results:

Gel image denoting sample numbers within each cycle number. Green arrow indicates the expected migration of our target band size of 166bp.

Looks like cycle 17 is the minimum cycle number with which we begin to see a consistent ~166bp band. Will continue on with the “prep-scale” PCR using 17 cycles.

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PCR – RLOv In-situ Hybridization (ISH) Probes

Ran probe-labeling PCRs to use in in-situ hybridization (ISH) using the PCR DIG Probe Sysnthesis Kit (Roche). Generated PCR probes for using the following BamHI-linearized plasmids:

  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_tail_fiber

The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).

Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.

All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.

Reaction calculations are here (Google Sheet): 20151109 – RLOv ISH Probe PCRs

Cycling params:

  1. 95C – 5mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 30s
  5. Go to Step 2, repeat 39 times.
  6. 72C – 10mins

After the PCR, 5μL of each reaction was run on a gel.

Results:

Hyperladder I (Bioline)

PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.

Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.

So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.

Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.

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Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

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PCR – RLOv Clones

Colony PCRs were performed on each of the transformations from 20151015 (RLOv_ DNA_helicase, RLOv_head_to_tail, RLOv_membrane_gene_1, RLOv_membrane_gene_2, RLOv_tail_to_fiber) to confirm successful ligations in plasmid pCR2.1 using the M13F/R vector primers.

Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp100+x-gal plate and then used to inoculate the respective PCR reactions.

Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.

Master mix calcs are here (Google Sheet): 20151019 – Colony PCRs RLOv

Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).

30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.

Results:

 

All the PCRs look good. All white colonies selected contain a PCR product of appropriate size (i.e. larger than the blue colonies; negative [-C] control). Will select clones #1 from each to grow up for plasmid prep.

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PCR – Oly RAD-seq Prep Scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.

After determining the minimum number of PCR cycles to run to generate a visible, 166bp band on a gel yesterday, ran a full library “prep scale” PCR.

 

REAGENT SINGLE REACTION (μL) x11
Template 40 NA
ILL-HT1 (1μM) 5 NA
ILL-BC# (1μM) 5 NA
NanoPure H2O 5 55
dNTPs (10mM) 20 220
ILL-LIB1 (10μM) 2 22
ILL-LIB2 (10μM) 2 22
5x Q5 Reaction Buffer 20 220
Q5 DNA Polymerase 1 11
TOTAL 100 550

 

Combined the following for PCR reactions:

  • 50μL PCR master mix
  • 40μL ligation mix
  • 5μL of ILL-HT1 (1μM)
  • 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.

NOTE: Samples 02, 03, & 04 did not have 40μL of the ligation reaction left (only 32μL) due to additional usage in the test scale PCR yesterday. Supplemented those three reactions with 8μL of H2O to bring them to 100μL.

 

SAMPLE BARCODE SEQUENCE
Oly RAD 02  1  CGTGAT
Oly RAD 03  2  ACATCG
Oly RAD 04  3  GCCTAA
Oly RAD 06  4  TGGTCA
Oly RAD 07  5  CACTGT
Oly RAD 08  6  ATTGGC
Oly RAD 14  7  GATCTG
Oly RAD 17  8  TCAAGT
Oly RAD 23  9  CTGATC
Oly RAD 30 10 AAGCTA

 

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
12 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

 

After cycling, added 16μL of 6x loading dye to each sample.

Due to limitations in available comb sizes and inability to combine combs to make larger well sizes, only loaded 58μL of samples in each well on this gel. Will load remainder on a second gel and combine after PCR products are purified.

Results:

 

Well, this is lame. There are absolutely no PCR products on this gel. In fact, this just looks like big smears of degraded DNA. I was expecting an amplicon of ~166bp to cut out of the gel. Based off of the test scale PCR from yesterday, everything should have been hunky dory. Not really sure what to think about this…

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PCR – Oly RAD-seq Test-scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.

Prior to generating full-blown libraries, we needed to run a “test-scale” PCR to identify the minimum number of cycles needed to produce the intended product size (166bp).

I ran PCR reactions on a subset (Sample #: 2, 3, & 4) of the 10 samples that I performed adaptor ligations on Friday.

PCR reactions were set up on ice in 0.5mL PCR tubes.

REAGENT SINGLE REACTION (μL) x4.4
Template 8 NA
NanoPure H2O 1 4.4
dNTPs (1mM) 4 17.6
ILL-LIB1 (10μM) 0.4 1.76
ILL-LIB2 (10μM) 0.4 1.76
ILL-HT1 (1μM) 1 4.4
ILL-BC1 (1μM) 1 4.4
5x Q5 Reaction Buffer 4 17.6
Q5 DNA Polymerase 0.2 0.88
TOTAL 20 52.8

 

Combined 12μL of master mix with 8μL of the ligation reaction from earlier today.

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
27 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

We’re following the “1/4 reduced representation” aspect of the protocol. As such, 5μL of each reaction was pulled immediately after the extension (72C – machine was paused) of cycles 12, 17, 22, & 27 in order to determine the ideal number of cycles to use. Also ran the ligation reactions (labeled “Ligations” on the gel below) of the samples as a pre-PCR comparison. Treated them the same as the PCR reactions: mixed 8μL of the ligation with 12μL of H2O, used 5μL of that mix to load on gel.

These samples were run on a 1x modified TAE 2% agarose gel (w/EtBr).

 

Results:

 

 

Test-scale PCR gel. Green arrow indicates desired band. The numbers below the headings indicate the sample number.

 

 

This looks pretty good. The green arrow on the gel indicates the desired band size (~166bp). Although difficult to see on this gel image, there is a gradient in band intensities across the cycles (band intensity increases as cycle number increases). Looks like we can use 12 cycles for our PCRs.

One other aspect of this gel that is very interesting is the ligations. The three ligation samples all show an intact high molecular weight band! This is very surprising, since the input gDNA from these three samples does not look this.

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PCR – RLOv for Cloning & Sequencing

After yesterday’s confirmation that the qPCR primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv, I needed to generate PCR products to clone and sequence.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

All samples were run in duplicate.

Master mix calcs are here: 20151009 – PCR RLOv

Cycling Params (PTC-200; MJ Research)

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 95
  • 600
40 Cycles
  • 95
  • 55
  • 72
  • 15
  • 15
  • 30

Samples were run on a 0.8% agarose 1x TBE gel, stained with ethidium bromide.

Results:

Amplification looks great. No amplification in no template controls (NTCs). Excised bands and purified products using Ultrafree DA Spin Columns (Millipore). Samples will be stored @ 4C until I am able to clone them for sequencing.

 

Gel image showing excised bands. And, it’s a complete hack job, which is embarrassing…

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Agarose Gel – Phage ISH Primers PCRs

Ran PCR products from yesterday on a 1% agarose 1x TBE gel, stained with ethidium bromide.

Results:

IMPORTANT NOTE: The negative control sample should actually be labelled UW08:22-11A.

 

PRIMER SET EXPECTED PCR SIZE (bp) RESULT SIZE (bp)
RLOv_membrane_gene_1 401 ~400bp
RLOv_membrane_gene_2 318 ~400bp
RLOv_tail_fiber_gene 451 ~500bp

PCR looks great. Excellent amplification in the RLO positive samples (06:6-54), with no amplification in the negative controls (UW08:22-11A) nor in the no template controls (NTC).

Excised the bands from each of the RLOv positive samples (see gel image below) and purified the DNA using UltrafreeDA Spin Columns (Millipore) according to the manufacturer’s protocol. DNA was stored @ 4C for cloning/labelling/sequencing at a later date.

Gel image showing excised regions.

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Agarose Gel – Geoduck & Olympia Oyster gDNA

Needed to assess the integrity of the newest gDNA isolated for the two genome sequencing projects: Geoduck gDNA from earlier today and Olympia oyster gDNA from 20151002.

Also needed to assess the integrity of the gDNA of ethanol-preserved Olympia oyster mantle tissue from Jake’s reciprocal transplant experiment, isolated on 20151002: samples NF1A & SN49A.

Ran samples on a 0.8% agarose, 1X modified TAE gel.

Loaded 1μL (~300ng) of the geoduck, Oly and NF1A samples.

Loaded 2μL (~100ng) of the SN49A sample.

Used 5μL of ladder.

Results:

 

Genome Sequencing Samples

The geoduck and the Oly samples look good. Intact, high molecular weight band. My only concern is the noticeable difference in band intensities between these two. Both samples should be ~300ng/μL, based on the NanoDrop1000 readings. However, it’s evident that the concentrations of these two samples differ greatly. Additionally, we can use the ladder to gauge the concentrations of the samples, since I loaded 0.5μg of the ladder, which is the quantity referenced on the ladder guide above.

It would appear that the geoduck sample concentration is closer to 60ng/μL (band intensity is similar to that of the 500, 1000, & 3000bp markers), as opposed to the 292ng/μL that the NanoDrop1000 indicated.

The Oly sample appears to have even less and appears less intense than the lowest concentration bands on the marker (16.0ng/μL). That’s not even remotely close to the 331ng/μL measured by the NanoDrop1000.

It’s difficult to say why this might be, as both samples were RNased and neither of them show extensive smearing (both of those factors would contribute to inflated spec readings).

Regardless, will ship them off to BGI to supplement the previous gDNA for this project.

Ethanol-Preserved Samples

Both samples show extensive smearing and no high molecular weight band, indicating they are both completely degraded. This is a very bad result for this project, as the tissue in this group is/was a bit of grasping at straws to obtain some intact DNA to use for the RAD-seq that we intend to pursue.

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