Tag Archives: PCR

Bisulfite NGS Library Prep – LSU C.virginica Oil Spill MBD Bisulfite DNA and Emma’s C.gigas Larvae OA Bisulfite DNA (continued from yesterday)

Continued library prep from yesterday. Set up Library Amplification according to the protocol. The samples received the following Barcode Indices:

  • HB2 – 1 (ATCACG)
  • HB5 – 2 (CGATGT)
  • HB16 – 3 (TTAGGC)
  • HB30 – 4 (TGACCA)
  • NB3 – 5 (ACAGTG)
  • NB6 – 6 (GCCAAT)
  • NB11 – 7 (CAGATC)
  • NB21 – 12 (CTTGTA)
  • 1A1 – 2 (CGATGT)
  • 1A2 – 1 (ATCACG)
  • 6A1 – 4 (TGACCA)
  • 6A2 – 5 (ACAGTG)
  • 103B1 – 6 (GCCAAT)
  • 103B2 – 7 (CAGATC)
  • 105A4 – 12 (CTTGTA)
  • 105A5 – 11 (GGCTAC)

Due to differences in input DNA quantities, samples were run with different numbers of thermal cycles.

13 thermal cycles were run for the following samples:

  • 1A1
  • 105A4
  • 105A5

22 thermal cycles were run for the following samples:

  • HB2
  • HB5
  • HB16
  • HB30
  • NB3
  • NB6
  • NB11
  • NB21
  • 1A2
  • 6A1
  • 6A2
  • 103B1
  • 103B2

Samples were quantified with 1uL of each sample using the Quant-iT dsDNA BR Kit (Invitrogen). Used 5uL of each standard and standards were run in duplicate.


PCR – Mac’s Bisulfite-Treated DNA

Realized that the PCR performed on 20140828 used the incorrect forward primer! As such, am repeating as before, but with the correct forward primer:

CgBS_733_26796F (SRID: 1597)

NOTE: Nothing left of sample EV2.28 bisulfite, so this was not run.


Ladder – O’GeneRuler 100bp Ladder (ThermoFisher)

Samples are loaded in numerical order from left to right, with a NTC sample before the second ladder.

All samples ran at ~275bp, which is larger than the previous gels. Confirmed with Mac that this gel looks correct. Will contact Cassie at Fred Hutchinson to go forward with PyroMark sequencing of these products.

Evidently, it would seem that Mac (and I) used the incorrect primer set when performing this PCR most recently. Doh!

PCR – Mac’s Bisulfite-Treated DNA

Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):

  • EV2.16 bisulfite
  • EV2.20 bisulfite
  • EV2.22 bisulfite
  • EV2.24 bisulfite
  • EV2.28 bisulfite
  • EV2.29 bisulfite
  • EV2.32 bisulfite
  • EV2.33 bisulfite

DNA needed to be diluted. Diluted according to this sheet provided by Mac:


NOTE: EV2.28 didn’t have sufficient DNA left to prepare the dilution according to Mac’s sheet. Instead, the remaining volume ofEv2.28 bisulfite DNA (0.5uL) was diluted in a total volume of 2.5uL to maintain the same dilution ratio.

Master mix calcs are here: 20140828 – PCR Mac Bisulfite Samples

Primers used were:

CgBS_733_26796Seq (SRID: 1598)
CgBS_733_26796R_5’biotin (SRID: 1596)

Cycling params:

  1. 1. 95C – 10mins
  2. 2. 94C – 30s
  3. 3. 56C – 30s
  4. 4. 72C – 30s
  5. 5. Repeat steps 2 – 5 44 more times
  6. 6. 72C – 10mins


Ladder used is O’GeneRuler 100bp DNA Ladder (ThermoFisher).

According to Mac, the expected band size is ~300bp. However, all samples are running at ~150bp. Mac is confused and does not know what to do.

*UPDATE 20140902* – Realized I used the wrong forward primer! Will repeat PCR with correct primer. Wonder if Mac did the same thing…

PCR – Hexokinase and Partial Exon #1

Performed PCR using newly designed primers to amplify the C. gigas hexokinase “promoter” (-2059bp from start) along with a portion of the first exon.

Primers used were Cg_Hk_Prom_pBAD_-2059 (SRID: 1518) and Cg_HK_Exon1_R (SRID: 1520).

Template used was C.gigas gDNA BB15 (from 20090519; 0.4216ug/uL). Master mix calcs are here. Cycling params are the same used on 20130227.

Samples were run in duplicate.


Lane 1: Hyperladder II (Bioline)

Lanes 2-3: C.gigas gDNA

Lanes 4-5: NTCs

We see a band of >2000bp (that’s the maximum on the molecular weight marker). The bands from each replicate were excised, purified using Ultrafree-DA columns (Millipore) and stored at 4C.

PCR – Hexokinase Promoter and CDS (repeat from 20130227)

Performed a repeat of the failed PCR from 20130227, but used a pool of cDNA (made from 20110311 C.gigas cDNA) instead of a single sample and changed the annealing temp to 50C.


Same exact results as 20130227; nothing. As such, didn’t take gel image. Will retry one more time using a long-distance polymerase, along with varying [MgCl2].

UPDATE 20130318: Doh! When talking about this at lab meeting today, I realized I’m trying to amplify the promoter (a genomic sequence) using cDNA! Will re-design primers and develop new cloning strategy for this!

PCR – Hexokinase Promoter and CDS

Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.

Used hexokinase primers (SR IDs: 1518, 1519).

Master mix calcs and cycling params are here.


Ladder: Hyperladder II (Bioline)

No amplification in sample or no-template control. Will re-do with lower annealing temp (50C).

PCR – COX/PGS Cloning Colony Screens from yesterday

Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:

Cg_COX1/2_qPCR_F (SR ID: 1192)

Cg_COX1_qPCR_R (SR ID: 1191)

Cg_COX2_454align1_R (SR ID: 1190)

Positive controls for both primers set were also run. The positive control template was the purified PCR product from 20111006.


Ladder is Hyperladder II (Bioline). Samples are loaded, left to right, as PGS1 and PGS2 on each colony (e.g. on the bottom gel image, under the “Colony 40″ label is the PGS1 rxn on the left and the PGS2 rxn on the right).

Nearly every colony exhibits amplification using both primer sets, w/the PGS1 reaction producing a band of ~250bp and the PGS2 reaction producing a band of ~750bp. Colonies 18 and 28 are an exception to this and produced no band with the PGS2 primer set. NTCs were clean. The positive controls worked as expected, yielding a band of ~250bp for PGS1 and a band of ~250bp for PGS2.

It is confusing as to why the size of the PGS2 positive control is different than the product that was generated from the colony PCRs.

Will select 10 colonies for mini-preps.

PCR – Purified COX/PGS 1/2 DNA from earlier today

Ran PCR using primers Cg_COX1/2_qPCR_F, Cg_COX1_qPCR_R, Cg_COX2_454align1_R (SR IDs: 1192, 1191, 1190; respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These reactions will verify (sort of) if we have both isoforms present in the PCR performed earlier today, prior to cloning. Master mix calcs and cycling params are here.


Lane 1: Hyperladder I (Bioline)

Lane 2: COX1/PGS1 primer set

Lane 3: COX1/PGS1 primer set NTC

Lane 4: COX2/PGS2 primer set

Lane 5: COX2/PGS2 primer set NTC

NTCs are clean for both primer sets. We see bands of the expected size for both primer sets. Additionally, we see lower expression in COX2/PGS2, as we observed in our previous qPCR reactions with these primer sets. Will clone the large fragment that was PCR’ed/purified from earlier today.