TITLE: Splinkerette Assay
AUTHORS: Aazam Vahdatshoar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol used to find the exact location of the integrated piggybac construct in the genome of stably transfected cell line. This assay determines the copy number of an exogenous gene in the established monoclonal piggybac cell  line as wells as their insertion loci.</div></div>

[STEPS]
?. [Harvest cells]
Passage and harvest the cells following the cell culture protocol.
100,000 cells would give enough genomic DNA.
?. [Harvest cells]
Pellet the cells, aspirate the supernatant, and transfer the pellet to a  Eppendorf tube.
1.5 mL
?. [Harvest cells]
Add  PBS and centrifuge at  for .
1 mL
Centrifuge: 400 34
?. [Harvest cells]
Remove the supernatant very carefully.
The pellet is very loose now.
?. [Harvest cells]
Snap freeze the pellet and store it at  for later genomic DNA extraction.
-80 °C
?. [Genomic DNA extraction]
Add  quick extract (QE) reagent to the pellet and vortex well. Aliquot QE reagent in  tubes and store at .
50 µl
1.5 mL
-20 °C
?. [Genomic DNA extraction]
Heat the tube of cell pellet in QE at  for .
65 °C
?. [Genomic DNA extraction]
Next, heat at  for .
98 °C
?. [Genomic DNA extraction]
Vortex well to dissolve the pellet completely.
?. [Genomic DNA extraction]
Measure the DNA concentration using a nanodrop.
?. [Sau3AI digestion]
Set all reactions from here to the end with a positive control alongside the gDNA prepared above.
You may use the piggybac plasmid construct that was transfected into the cells for positive control.Genomic DNA 2 µgSau3AI enzyme (5-20 units) 1 µL10X cutsmart buffer 3 µLDNase/RNase-free H2O to final volume 30 µLSet the digestion reaction as listed in a   tube.
?. [Sau3AI digestion]
Incubate at  in an incubator or heat block .
37 °C
?. [Sau3AI digestion]
Heat inactivate the reaction at  for .
65 °C
?. [Sau3AI digestion]
Purify Sau3AI digested gDNA using PCR purification kit following manufacturer protocol. Elute DNA in  H2O.
30 µl
?. [Sau3AI digestion]
Store the tube at  for later.
-20 °C
?. [Ligation of adaptors]
Sau3A1-digested gDNA (from step 6.3) 300 ngAdaptor mix (25 µM) (prepared above) 1 µL10X T4 DNA ligase buffer 4 µLT4 ligase 1 µLDNase/RNase-free H2O to final volume 40 µL
?. [EcoRV digestion]
Ligation reaction (from step 6.4) 40 µLEcoRV enzyme 1 µL10X cutsmart buffer 10 µLDNase/RNase-free H2O 49 µLFinal volume 100 µLSet the digestion reaction as listed in a   tube.
?. [EcoRV digestion]
Incubate at  in an incubator or heat block .
37 °C
?. [EcoRV digestion]
Purify EcoRV digestion reaction using PCR purification kit following manufacturer protocol. Elute DNA in  TE buffer.
32 µl
?. [EcoRV digestion]
Store the tube at  for later.
-20 °C
?. [Spkt1 PCR]
Set up the primary PCR amplification in a PCR tube.Taq 2X Master Mix 10 µLSpkt-Primer Mix-1 1.5 µLTemplate (from step 6.5.3 elute) 5 µLDNase/RNase-free H2O 3.5 µLFinal volume 20 µL
Taq 2X Master Mix 10 µLSpkt-Primer Mix-1 1.5 µLTemplate (from step 6.5.3 elute) 5 µLDNase/RNase-free H2O 3.5 µLFinal volume 20 µL
?. [Spkt1 PCR]
Run the reaction in the thermal cycler with the following program for Taq 2X master mix polymerase.Initial denaturation 95°C 3 min 1XDenaturation 95°C 30 sec34XAnnealing 55°C 30 sec Extension (1min/kb) 68°C 2 minFinal extension 68°C 5 min 1XHold 4°C Forever
Initial denaturation 95°C 3 min 1XDenaturation 95°C 30 sec34XAnnealing 55°C 30 sec Extension (1min/kb) 68°C 2 minFinal extension 68°C 5 min 1XHold 4°C Forever
?. [Spkt1 PCR]
Store PCR product at .
-20 °C
?. [Spkt2 nested PCR]
Set up the secondary nested PCR in a PCR tube.Taq 2X Master Mix 10 µLSpkt-Primer Mix-2 1.5 µLTemplate (from step 6.6.3) 1 µLDNase/RNase-free H2O 7.5 µLFinal volume 20 µL
Taq 2X Master Mix 10 µLSpkt-Primer Mix-2 1.5 µLTemplate (from step 6.6.3) 1 µLDNase/RNase-free H2O 7.5 µLFinal volume 20 µL
?. [Spkt2 nested PCR]
Run the reaction in the thermal cycler with the following program for Taq 2X master mix polymerase.
Annealing temperature and final extension time are different from the 1st PCR.Initial denaturation 95°C 3 min 1XDenaturation 95°C 30 sec34XAnnealing 57°C 30 sec Extension (1min/kb) 68°C 2 minFinal extension 68°C 20 min 1XHold 4°C Forever
?. [Spkt2 nested PCR]
Store PCR product  at .
-20 °C
?. [Topo TA cloning and transformation]
Run the PCR product from step 28 on 1.5% agarose gel.
The number of bands would indicate the number of integration sites if more than one copy is integrated. It may also indicate the polyclonal nature of the cell line with different integration locus in each clone. The positive control plasmid should show only one band.
?. [Topo TA cloning and transformation]
Gel purify each band separately. Store at .
-20 °C
However, fresh product would be more efficient for Topo TA cloning.
?. [Topo TA cloning and transformation]
Set up ligation reaction in PCR tube as following:Gel-purified PCR product (from step 6.8.2) 0.8 µL (0.4-1.6 µL)Salt solution 0.4 µLTopo vector 0.4 µLDNase/RNase-free H2O 0.8 µLFinal volume 2.4 µL
Gel-purified PCR product (from step 6.8.2) 0.8 µL (0.4-1.6 µL)Salt solution 0.4 µLTopo vector 0.4 µLDNase/RNase-free H2O 0.8 µLFinal volume 2.4 µL
?. [Topo TA cloning and transformation]
Incubate the reaction at  for less than .
-20 Room temperature
?. [Topo TA cloning and transformation]
Transfer to ice or store at  for later use.
-20 °C
?. [Topo TA cloning and transformation]
Mix gently  ligation reaction with  competent cells in  tube . Incubate  for .
1 µl
20 µl
1.5 mL
on ice
on ice
?. [Topo TA cloning and transformation]
Heat shock at  for . Leave  for -.
42 °C
on ice
?. [Topo TA cloning and transformation]
Add  SOC medium to the transformed cells. Incubate at  in an orbit shaker for .
200 µl
42 Room temperature
37 °C
?. [Topo TA cloning and transformation]
Plate  and  on pre-warmed agar plate supplemented with  ampicillin. Incubate at .
20 µl
100 µl
37 °C
?. [Topo TA cloning and transformation]
Pick a few colonies from each plate. Inoculate into  LB broth supplemented with  ampicillin. Incubate at  in an orbit shaker .
5 mL
37 °C
?. [Topo TA cloning and transformation]
Extract the DNA using Qiagen DNA miniprep.
?. [Sequencing]
Provide  volume of plasmid prep from step 39 in PCR tube. The final concentration needs to be -.
Topo TA cloning kit uses vector pCR4 for cloning. Primers M13 reverse and M21 forward have binding sites on either side of the cloning site on the vector. We only need to sequence with one of these primers. Though the primers are provided in the TOPO TA cloning kit you may submit the samples without them because sequencing facilities have these primers, too. Follow the guidelines on submitting samples for sequencing provided on vendor’s website.
10 µl
?. [Sequencing]
Fill the submission form on vendor’s website. Choose their relevant in-house primer from their primer list and mention on the form.
The sequencing result would show the genomic sequence right before the 5‘ ITR end of the piggybac construct as well as part of the construct.
?. [Sequencing]
Blast the genomic sequence on NCBI website for the exact locus the piggybac construct has integrated.
?. [Ligation of adaptors]
Incubate the ligation reaction at  (12 to 16 hours).
4 °C
?. [Ligation of adaptors]
Heat inactivate the reaction at  for .
65 °C
?. [Ligation of adaptors]
Cool down the sample to .
65 Room temperature