Tag Archives: ligation

Adaptor Ligation – Oly AlfI-Digested gDNA for RAD-seq

Continued to follow the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Digested DNA from yesterday was heat inactivated for 10mins @ 65C and was not run out on a gel due to the fact that the input gDNA was degraded and a shift in the high molecular weight band (indicating the digestion was successful) would not exist because a high molecular weight band is absent in these samples.

 

Anneal Adaptors

After preparing the two adaptors below, they were incubated for 10mins @ RT:

  • Adaptor 1 (2μM final concentration of each oligo): 1.5μL of 5ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total
  • Adaptor 2 (2μM final concentration of each oligo): 1.5μL of 3ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total

After annealing, the adaptors were stored on ice.

 

Adaptor Ligation

All components were stored on ice. Ligation reactions were prepared on ice and performed in 0.5mL snap cap tubes.

REAGENT SINGLE REACTION (μL) x11
Digested DNA 10 NA
ATP (10mM) 1 11
10x T4 Ligase Buffer 4 44
Adaptor 1 (2μM) 5 55
Adaptor 2 (2μM) 5 55
T4 DNA Ligase 1 11
NanoPure H2O 24 264
TOTAL 50 440

Combined 40μL of the master mix with 10μL of AlfI-digested DNA in a 0.5mL snap cap tube.

Incubated ligation reaction @ 16C O/N in PTC-200 thermal cycler (MJ Research) – no heated lid.

Ligations will be stored @ -20C until I can continue working with them on Tuesday.

Share

Adaptor Ligation – Oly AlfI-Digested gDNA for RAD-seq

Continued to follow the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Digested DNA from earlier today was not run out on a gel due to the fact that the input gDNA was degraded and a shift in the high molecular weight band (indicating the digestion was successful) would not exist because a high molecular weight band is absent in these samples.

 

Anneal Adaptors

After preparing the two adaptors below, they were incubated for 10mins @ RT:

  • Adaptor 1 (2μM final concentration of each oligo): 1.5μL of 5ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total
  • Adaptor 2 (2μM final concentration of each oligo): 1.5μL of 3ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total

After annealing, the adaptors were stored on ice.

 

Adaptor Ligation

All components were stored on ice. Ligation reactions were prepared on ice and performed in 0.5mL snap cap tubes.

REAGENT SINGLE REACTION (μL) x11
Digested DNA 10 NA
ATP (10mM) 1 11
10x T4 Ligase Buffer 4 44
Adaptor 1 (2μM) 5 55
Adaptor 2 (2μM) 5 55
T4 DNA Ligase 1 11
NanoPure H2O 24 264
TOTAL 50 440

Combined 40μL of the master mix with 10μL of AlfI-digested DNA in a 0.5mL snap cap tube.

Incubated ligation reaction @ 16C for 3hrs in PTC-200 thermal cycler (MJ Research) – no heated lid.

Ligations were stored @ -20C until I can continue working with them on Monday.

Share

Adaptor Ligation – Oly AlfI-Digested gDNA for RAD-seq

Yesterday’s AlfI over night restriction digest was heat inactivated by heating @ 65C for 10mins. Samples were stored on ice.

Continued to follow  the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Digested DNA was not run out on a gel due to the fact that the input gDNA was degraded and a shift in the high molecular weight band (indicating the digestion was successful) would not exist because a high molecular weight band is absent in these samples.

The following oligos were reconstituted in TE buffer (pH = 8.0) to 100μM:

  • 3ILL-NR
  • 5ILL-NR
  • anti-ILL
  • ILL-BC1 (Barcode sequence: CGTGAT)
  • ILL-HT1 (Barcode sequence: ATGCAT)
  • ILL-HT2 (Barcode sequence: CGTACG)
  • ILL-LIB1
  • ILL-LIB2

Anneal Adaptors

After preparing the two adaptors below, they were incubated for 10mins @ RT:

  • Adaptor 1 (2μM final concentration of each oligo): 1.5μL of 5ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total
  • Adaptor 2 (2μM final concentration of each oligo): 1.5μL of 3ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total

After annealing, the adaptors were stored on ice.

 

Adaptor Ligation

All components were stored on ice. Ligation reactions were prepared on ice and performed in 0.5mL snap cap tubes.

REAGENT SINGLE REACTION (μL) x11
Digested DNA 10 NA
ATP (10mM) 1 11
10x T4 Ligase Buffer 4 44
Adaptor 1 (2μM) 5 55
Adaptor 2 (2μM) 5 55
T4 DNA Ligase 1 11
NanoPure H2O 24 264
TOTAL 50 440

Added 40μL of the master mix to each tube of AlfI-digested DNA (12μL). NOTE: I made a mistake here. I should have only combined 10μL of DNA with the 40μL of master mix for each. My mistake was due, in part, to the way the Meyer Lab 2bRAD protocol is written. In the Digestion section of the protocol, Step 5 (run 2μL of the digests on a gel) is listed as optional. However, in Step 2a of the Ligation section, it says to add the “remaining 10μL of digested DNA”. The use of the word “remaining” in this instance is misleading because it implies to use all that’s left in the tube.

Incubated ligation reaction @ 16C for 3hrs in PTC-200 thermal cycler (MJ Research) – no heated lid.

Transferred tubes to ice while preparing subsequent

Share

Cloning – C.gigas COX2/PGS2 5’/3′ RACE Products (from earlier today)

The bands that were excised and purified earlier today were cloned in to pCR2.1 using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol with the following changes: used 4uL of all PCR products, incubated ligation reactions for 15mins @ RT, incubated competent cells with ligation reactions for 15mins on ice.

Two volumes of each reaction were plated (50uL and 100uL) on Kan50 plates with X-gal (made 20010412 by SJW) and incubated @ 37C O/N.

Results:

Ample number of white colonies for all 4 sets of cloning reactions.

Share

Ligations – COX1/COX2 PCR Products

Performed ligations/cloning on a variety of COX1 genomic and COX 5′ RACE products using the TOPO TA Cloning Kit (Invitrogen). Used 2uL of gel-purified PCR product in each cloning rxn, 2.5uL of H2O, 1uL of salt solution, and 0.5uL of the Invitrogen pCR2.1 vector. Incubated samples for 5mins at RT. Used 2uL of the cloning reaction to transform TOP10 chemically competent cells (Invitrogen), mixed very gently, incubated on ice for 5mins, heat shocked at 42C for 30s and immediately placed on ice. Added 250uL of SOC Medium and incubated tubes at 37C, 200RPM for 1hr. Plated 50uL of each transformation on LB+Kan plates (made by Steven on unknown date with unknown Kan concentration) containing 40uL of 40mg/mL X-gal. Incubated O/N at 37C. Remaining volume of transformed bacteria were stored @ 4C.

Share

Hibridizaton/Ligation SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

All 8 samples were hybridized/ligated according to Ambion’s Whole Transcriptome Analysis Kit using Adaptor A.

Share

RNA Adapter Hybridization and Ligation – Herring Liver mRNA for SOLiD Libraries

RNA from yesterday was speedvac’d to dryness and resuspended in 3uL of nuclease-free H2O. Samples were mixed with Adaptor Mix A and hybridized according to Ambion WTK protocol. Samples were then ligated for 16hrs @ 16C, according to Ambion WTK protocol.

Share

Reverse Transcription/cDNA purification/Emulsion PCR – Ligation rxns of trout fragmented RNA for SOLiD WTK (from yesterday)

The four samples from yesterday were prepared according to the Agilent SOLiD WTK protocol. Briefly:

 

 

Results: All four samples appear to have cDNA. Interestingly, the “Amped cDNA trout RBC control ribo(-)” sample was the sample that had no detectable RNA after fragmentation, BUT this sample produced the highest yield of cDNA… See below.

1.5uL of each sample was transferred to a 0.5mL snap cap tube and stored @ -80C in the “Samples for Bioanalyzer” box for submission on the DNA 1000 Chip.

The Yellow/Brown plot above is the “Amped cDNA trout RBC poly I:C ribo(-) & polyA” sample and exhibits a strange profile at the 220-230nm range that differs than the three other samples.

Share

Adapter Ligation – Rick’s trout fragmented control/poly I:C samples for SOLiD WTK

See the Next Gen Seq Library Database for more info. Processed the 4 samples (one set Ribominus only, one set Ribominus + PolyA enriched) according to the Agilent WTK. Briefly:

  • Speedvac’d samples to dryness
  • Resuspended RNA in 3uL H2O
  • Adapter rxn. Used all 3uL of RNA (used only 1uL of RBC Ribo only sample due to high concentration)
  • Ligation rxn

Incubated 16C for 16hrs.

Share