Rad Sequence
The text below is a partial copy of Ovidiu's protocol with my notes after watching Petra run it on 21 Oct 2020.
IMPORTANT! The first part of the protocol has to be done in two consecutives days because day 1 ends with "incubate at 16°C overnight". After that the protocol can be stopped after each cleaning process and resumed days later.
Water used for elution during the whole protocol is from Merck
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Day 1
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Petra did this part on Tuesday, when I am not at the Institute. According to her this is the easiest part of the whole protocol. I was here for day 2 and 3 (Wed and Thursday)
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Day 2
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Turn on the Bioruptor/sonicator (room 225) to start cooling the water
4. Pool Samples, making sure each sample has a different P1 adapter.
Pool such that you have 1-2 μg (max. 10 μg) to shear - so you have enough template DNA surviving until the end of the protocol.
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5. Bioruptor
note: It's possible to sonicate one or two samples and then check them on the bioanalyzer but the chip costs ca. 50 Euros.
Fill up the log sheet at the door (Room 225)
Use 100 μl DNA in each 0,65 ml Bioruptor tube. Air bubbles must be avoided!
note: different quantities might give different results. Petra sheared two samples, one with 100μl and one with 60 μl. Results on the Bioanalyzer were quite different which lead Petra to discard the sample with 60μl.
For Dactylorhiza 3 cycles of 30s on /60s off.
For Heliosperma 2 cycles of 45s on/60s off. (Petra used this one for Allium)
note: After shearing, combine the samples back if you had to aliquote them for the sonicator.
Clean with a Qiagen MiniElute Reaction or PCR clean up column.
Note: Petra used the MiniElute Reaction at this step and the PCR clean up for the remaining of the protocol
For the Qiagen MiniElute Reaction use the Quick Start Protocol that comes with it.
Add 300μl of the Buffer ERC even if you have more than 100μl of sample (e.g., 700μl of sample)
This Qiagen protocol gets rid of fragments <70bp and >4Kb.
(note: step 8: elution is done with water.)
Elute in up to 100 μl per pool. (note: Petra does 50μl twice)
(note: Elution volume was 100μl (2 times 50μl) instead of 10μl indicated on Qiagen Protocol)
Double-size select with SPRI Based Size Selection
User guide PDF (Whole or Partial)
(note:Right and Left refer to fragments smaller and larger than the desired size. )
0.55x on the right & 0.7x on the left.
(note: Petra had 97 μL. For the left she added (0.8-0.55=) 0.25 μL instead of 0.7μL from Ovidiu's protocol.)
Elute the DNA in 20 μl ddH2O.
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6. Quick Blunting
Use the Quick Blunting Kit (NEB) to polish the ends of the DNA
| per sample | # of pools + 0.5 | total for the MM |
|---|---|---|
| 19 μl DNA | ||
| 2.5 μl Buffer | ||
| 2.5 μl dNTP | ||
| 1.0 μl Enzyme |
The 0.5 on the table above is just to make sure you will have enough MM.
Room temperature, 30 minutes!
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Purify with Qiagen MinElute Reaction or PCR Cleanup Kit, elute in 16 μl water
(note: Petra used the PCR Cleanup kit)
Samples can now stay at -20°C for a few days
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Day 3 (was done in lab 204 to avoid many people on the third floor lab)
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7. Add dATP
| #pools+0.5 | MM | |
|---|---|---|
| 2 Klenow exo- (15U) | ||
| 1 μl dATP (100mM) | ||
| 2 μl Buffer 2 NEB | ||
| 15 μl DNA | do not add to the MM |
Incubate at 37°C for 30 min.
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Purify with Qiagen Reaction or PCR Cleanup Kit, elute in 22 μl water.
note: Petra uses Qiagen MinElute PCR Cleanup Kit.
Follow the protocol (web site or PDF:page 17) that comes with the kit except for the final elution, use 22 μl of water!
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Quantify with PicoGreen or Qubit and use in the next step the same amount for each sub-library! (i.e., normalize to the lowest of the sublibraries)
We used the Qubit.
Settings: Double Strand DNA > High sensitivity
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Make a table with the Qubit values then bring down the concentration of all samples to the lowest concentration measured. A sample table from Petra, with adaptor combinations and of the calculation of sample-volumes for P2 can be found here: see P2-calculation.
E.g., On the table below, the lowest concentration was 9.6, so all other samples had to be diluted to also have 9.6 ng/ul.
8. Ligate P2 Adapter
Which adapter goes with which DNA should be written down. Petra used this table.
For 6 pools use 6 x 1.5 mL tubes. To each tube add MM (4.5ul) + P2 adapter and DNA (already adjusted to the lowest measured concentration - seen on previous step).
| Reagents | x (number of samples) + 0.5 | amount in Master Mix |
|---|---|---|
1ul 100mM rATP | 6.5 | 6.5 ul |
| 3 ul NEB buffer 2 | 6.5 | 19.5 ul |
| 0.5 ul T4 ligase | 6.5 | 3.25 ul |
| 5ul P2 Adapter (2uM) | do not add to MM | |
| 20.5 ul DNA | do not add to MM |
P2 Adapter [2 μM] 5 μl
100mM rATP 1μl
NEB buffer 2 3 μl
T4 Ligase (2000000U) 0.5 μl
DNA 20.5 μl
Total 30 μl
Room temperature for 30 minutes.
Pool all samples (now they can be distinguished by different P1 and P2 combinations)
SPRI size selection – only left side 0.7 x, elute in 50μl water
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9. PCR
Perform 6x50 μl PCR reactions to generate enough templates for sequencing. Keep 5ul of a “positive control” which is not going into the PCR machine (but contains DNA as well).
DNA 2 μl
2x Phusion Master Mix 25 μl
P1-PCR primer (100uM) 0.2 μl
P2-PCR primer (100uM) 0.2 μl
Water 23 μl
TOTAL 50.4 μl
98ºC 30 seconds
98ºC 10 seconds
65ºC 30 seconds 18 x
72ºC 30 seconds
then
72ºC 5 min
4ºC hold
10. Check 4 ul of 2-3 PCR products on a 1% TBE gel. Load the same amount (i.e., 4ul) of the positive control in a lane as well. The positive control should be at least 2x less visible.
RAD PCR products (1.2% agarose, 0.5% TBE)
Lanes 1-5 are RAD PCR products (25 μl each, for 25 cycles)
Lane 6 is the negative control
Lane 7 is a 25 μl PCR cleaned up with a Qiagen spin column (primer-dimer remains). This photos does not contain a positive control!
Note for the Bioanalyzer
At the Bioanalyzer change the essay before entering sample names otherwise they will be deleted if the essay is changed.
Do not vortex/spin the gel (blue + red reagents combined). Follow instructions on the Quick Start Guide of the Bioanalyzer. There is one for DNA and one for RNA (depending on what you are working with)
obs: on the protocol, if not using all the wells of the chip, then load 6μl of MARKER instead of the 5μl to compensate for the 1μl of sample that won't be loaded. This is actually mentioned in the Guide.