TITLE: Prepare Samples for Miseq
AUTHORS: FishFloorUCL

[DESCRIPTION]
(MiSeq nano V2 chip 300 cycle), MiSeq multiplexes sequences, allowing you to DNA sequence multiple samples at once. This is particularly useful for genotyping fish with mosaic genotypes, such as F0 knockouts. 

@FishFloorUCL

[STEPS]
SECTION: Design Miseq Primers
1. Design primers surrounding your site of interest

Optimal amplicon size 200bp
As far as possible, the site of interest should be in the middle of the designed amplicon. (This is so that it has the best chance of being read by both the forward and reverse reads)
If possible, try and avoid designing primers in non-coding regions of DNA since these regions are more likely to have variation and SNPs, which might affect the efficacy of your primer.

I usually use the NCBI Primer-Blast tool. Enter ~300bp of DNA sequence around your region of interest under 'PCR Template' and set the 'Range' for the forward and reverse primer so that your site of interest is more or less in the middle of the PCR product.  

If you previously used a set of primers to check your TALLEN/CRISPR efficiency using Melt Curve Analysis, or if you have your primer sequence from CHOPCHOP,  you can use the same sequence to design the primers for MiSeq. Anecdotally this seems to work well for most people on their first try.
SECTION: Design Miseq Primers
2. Add miseq tags/overhangs to your primer sequence.

Universal forward (F) tag: 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG 3'
Universal reverse (R) tag: 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG 3'

  
These overhang sequences correspond to the Illumina nextera transposase overhangs and will be compatible with Illumina kits using Nextera XT
SECTION: Design Miseq Primers
3. (Optional) Test primers (including tag) for secondary structure formation (e.g., dimers and hairpins) using this online IDT OligoAnalyzer tool

NB. It is unlikely that you will have high scores due to the length of the primers and amplicon restrictions. Just pick the best 
SECTION: Design Miseq Primers
4. Order primers - standard desalted primers are fine for this application.
SECTION: (Optional) Test Primers
6. 
 


Run a PCR
I have designed a handy master mix calculator for Taq, Platinum Taq and Pre-mix Schier PCR mix. 
Here is an example of a PCR mix for a single well with a 10 µL total volume
     Autoclaved, distilled (nuclease free) water      6.46 μL        10X PCR Buffer, -Mg      1 μL        50 mM MgCl2      0.3 μL        10 mM (each) dNTP Mix      0.2 μL        forward primer      0.5 μL        reverse primer      0.5 μL        Taq DNA Polymerase      0.04 μL        Template DNA      1 μL    
Note, Taq is unstable and should be taken out of the freezer for the shortest amount of time possible. It is sensible to prepare PCR mix on ice.  

Suggested PCR programme:
95 °C , 5 min 
95 °C , 30 s (Denaturation step)
Temperature gradient* around 60 °C , 30 s (Annealing step)
72 °C , 30 s (Extension step)
Repeat steps 2-4, 25-35 times 
72 °C , 5 min (Final extension step)
10 °C hold

*For your test run, you should use a gradient PCR to test the ideal temperature for your primers (Note. By default, CHOPCHOP designs primers for an optimal melting temperature of 60°C). We usually run a gradient PCR for five different temperatures, with 60°C being optimal, then varying 1°C-1.5°C above and below.
SECTION: Prepare samples for miseq
8. Amplify up PCR products to analyse by MiSeq.

 

I have designed a handy master mix calculator.
Here is an example of a PCR mix for a single well with 10 µL total volume 
          Autoclaved, distilled (or nuclease free) water----20 μL40 μL0.7710X PCR Buffer, -Mg10X1X2.5 μL5 μL0.1150 mM MgCl250mM1.5mM0.75 μL1.5 μL0.030.310 mM (each) dNTP Mix10mM0.2mM0.5 μL1 μL0.020.2forward primer10μM0.2μM0.5 μL1 μL0.020.2reverse primer10μM0.2μM0.5 μL1 μL0.020.2Platinium Taq DNA Polymerase5U/μL1.25U/rxn0.25 μL0.5 μL0.010.1Template DNAvaries *< 500ng/rxn2.550.11
Note, Taq is unstable and should be taken out of the freezer for the shortest amount of time possible. It is sensible to prepare PCR mix on ice. 

Suggested PCR programme:
95 °C , 5 min (Initial Denaturation Step)
95 °C , 30 s (Denaturation step)
60 °C* 30 s (Annealing step)
72 °C , 30 s (Extension step)
Repeat steps 2-4, 25-35 times 
72 °C , 5 min (Final extension step)
10 °C hold

*Or Tm of your given primer pair
SECTION: Design Miseq Primers
5. If the primers arrive as dry pellets, reconstitute the primers in autoclaved distilled or nuclease-free water to create a stock solution and working solution.
100 micromolar (µM) stock solution by adding nuclease-free water. If    nMoles of your primer (check the tube), resuspend the dry pellet in   µL of water 
10 micromolar (µM) working solution
SECTION: (Optional) Test Primers
7. Run on a 1% agarose gel
 

SECTION: Prepare samples for miseq
9. Run on a 1% gel to confirm a single band at the anticipated weight. This is just to check the PCR worked. To save time, just run a couple of your samples.
SECTION: Prepare samples for miseq
11. Check the concentration of your clean PCR product. This can be done using Nanodrop or Qubit. I would recommend Qubit due to its superior accuracy and reliability. We recommend only testing a few of your samples to save time and money.

We usually use the following kit from Thermo Fisher, which comes with a working solution.  
  

Setup and label the lids of the required number of assay tubes for your samples plus two standards (Use thin-wall, clear, 0.5-mL Qubit Assay PCR tubes (Cat. No. Q32856)). 
Add 190 µL Qubit™ 1X dsDNA working solution to each standard tube
Add 10 µL of each Qubit standard to the appropriate standard tube
Add 199 µL Qubit™ 1X dsDNA working solution to each sample tube
Add 1 µL of each sample to the appropriate tube
All tubes should have a final volume of 200 µL 

 
Read concentration using the Qubit machine and note them down. 
SECTION: Prepare samples for miseq
10. Clean up your samples

Traditional column-based PCR clean-up kits would work fine, but we recommend using ExoSAP,   . Although it is more expensive (~£30 per plate), it is much more practical, especially for many samples. 

Transfer 5 µL of your PCR product into a new plate 
Add 2 µL  of ExoSAP reagent to each well
(Vortex optional) Spin down & run the ExoSAP programme thermocycler.
15 min at 37 °C – Degrade the remaining primers and nucleotides 
15 min at 80 °C – Inactivate ExoSAP
SECTION: Prepare samples for miseq
12. Prepare your samples for miseq:
You need to submit a minimum of 6 µL at a final concentration of 15-25 ng/μL
 
Label your plate/tubes with your name, the date and 'For Miseq' and leave the samples with the Payne lab in the Cancer Insititute or in the Fish Facility B03 fridge in the anatomy building, whichever is most convenient for you. Once you have dropped off your samples, email Alexandra Lubin, letting her know.