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tags: ebook
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# Amplicon Barcoding - 16s from bacteria

## Members
* José Agosto Rivera
* Miguel Urdaneta Colón
* Cristina Andújar Sierra
* Miguel A. Aguilar
* Gene Espinoza
* Luis E. Vázquez Quiñones
* Jeremy Seto

## First part of the workflow

### First PCR Preparation
* The first PCR use primers that amplify the target sequence & add an anchor sequence. The anchor sequences at the 5'-end serve as priming sites for barcoded outer primers used in the 2^nd PCR. The V1-V9 region (V stands for variable) of the 16S rRNA gene is amplified using the inner primers listed below.
*
* **Forward**
* 5'-TTTCTGTTGGTGCTGATATTGC-**target-specific sequence**-3'

* **27F**
* 5'-TTTCTGTTGGTGCTGATATTGC**AGRGTTYGATYMTGGCTCAG**-3'
* **Reverse**
* 5'-ACTTGCCTGTCGCTCTATCTTC-**target-specific sequence**-3'

* **1492R**
* 5'-ACTTGCCTGTCGCTCTATCTTC**CGGYTACCTTGTTACGACTT**-3'

### **Thermal Cycler Program**

|**Step**|**Temperature**|**Time**|**Cycles**|
|:--|:--:|:--:|:--:|
|Initial Denaturation|95°C|3 minutes|1|
|Denaturation|95°C|15 seconds|25-35 |
|Annealing|55°C|15 seconds|^ |
|Extension|72°C|30 seconds|^ |
|Hold | 4°C|∞| |

## Second part of the workflow
[Protocol: cDNA PCR barcoding sequencing](https://store.nanoporetech.com/us/cdna-pcr-barcoding-sequencing-kit-1.html)

### Second PCR Preparation to add the barcodes:

* Materials and Reagents:
* - [ ] Pippetes
* - [ ] Tips
* - [ ] Tubes
* - [ ] Sample
* - [ ] Nuclease Free Water
* - [ ] Barcoding Primers
* Steps:
1. Dilution Preparations: 1:10 & 1:100
* 1:10 (B)
* - [ ] 1 µL Sample for the 1st PCR
* - [ ] +9 µL Nuclease Free Water
<br>
* 1:100 (C )
* - [ ] 1 µL Sample B
* - [ ] 9 µL Nuclease Free Water
* - [ ] Add 1 µL Barcode Primer to Samples B & C
* Total amount in Sample B and C should be 11 µL

2. PCR Sample Preparation:
* In other tube mix:
* - [ ] 5 µL of B Sample (1:10)
* - [ ] 6.75 µL Nuclease Free Water (verify 7.5 µL for the future)
* - [ ] 12.5 µL Master Mix
* - [ ] Do the same mix using the another tube with Sample C
* - [ ] Mix the slution gently by pipetting.
* - [ ] Your Samples are ready yor the PCR

### PCR Thermal cycler program for adding the barcodes by PCR


### Electrophoresis
* Materials
* - [ ] Gel casting tray
* - [ ] Agarose
* - [ ] SYBR safe
* - [ ] Electrophoresis Machine
* - [ ] Comb
* - [ ] Parafilm
* Steps:
* Prepare the mix for each sample
* - [ ] 5 µL sample + 1 µL loading dye 6X
* We do it with both of samples
* - [ ] Cut a piece of parafilm and place 1 µL of dye separated for the amount of samples that are available.
* - [ ] Mix the Sample with the loading dye
* - [ ] Two gels are needed one for dilution 1:10 and another for dilution 1:100.
* - [ ] We run it with 150 voltage for 15 minutes
* - [ ] When the run finish we can see our result in a UV light
* - [ ] Analys the Electrophoresis result and select the best dilution for your work (In our situation we select sample C)

### PCR DNA Cleanup

* Steps
* - [ ] Transfer each sample to a clean 1.5 mL Eppendorf DNA LoBind tube.
* - [ ] Resuspend the AMPure XP beads by vortexing.
* - [ ] Add 20 μL of resuspended AMPure XP beads to each 1.5 mL Eppendorf DNA LoBind tube.
* - [ ] Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
* - [ ] Prepare 5 mL of fresh 70% ethanol in nuclease-free water.
* - [ ] Spin down the samples and pellet on a magnet. Keep the tubes on the magnet, and pipette off the supernatant.
* - [ ] Keep the tubes on the magnet and wash the beads with 200 μL of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
* - [ ] Repeat the previous step.
* - [ ] Spin down and place the tubes back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds,but do not dry the pellets to the point of cracking.
* - [ ] Remove the tubes from the magnetic rack and resuspend each pellet in 12 μL of Elution Buffer (EB).
* - [ ] Incubate at room temperature for 10 minutes.
* - [ ] Pellet the beads on the magnet until the eluate is clear and colourless.
* - [ ] Remove and retain 12 μL of each eluate into a clean 1.5 mL Eppendorf DNA LoBind tube.

### DNA Quantitation:
#### Use the Qubit to determine the [DNA]
* Materials
* - [ ] Invitrogen Qubit Assay Tubes Catalog number: Q32856 0.5 mL PCR microtubes thin-wall, clear or Axygen PCR-05-C tubes (VWR, part no. 10011-830)
* - [ ] Qubit dsDNA HS Reagent
* - [ ] Qubit dsDNA HS Buffer
* - [ ] Qubit dilution buffer
* - [ ] Qubit instrument
* - [ ] P-1000 & tips
* - [ ] P-100 or P-200 & tips
* - [ ] P-10 & tips
* - [ ] Disposal container for the tips
<br>
* Steps to quantify the DNA
- [ ] 1. Set aside two microtubes for the standards (dsDNA High Sensitivity needs 2 standards) and one microtube per each sample.
- [ ] 2. Label the microtubes on the lid (DO NOT label on the side), the instrument will be reading the sample through the microtube side.
- [ ] 3. The working solution needs to be prepared in a plastic tube diluting the dsDNA HS reagent 1:200 in dsDNA HS Buffer.
- [ ] a. In our case we have 10 samples (Diaprepes 6 & RBL 4) & 2 standards.
- [ ] b. Now use your Qubit reagent calculator and indicate that you have 10 samples & 2 standards.
- [ ] c. Prepare the Qubit working solution by mixing 13 µL (= 1/200 x 2,600 µL) of the Qubit dye & 2,587 µL (= 2,600 µL - 13 µL) Qubit dilution for a total volume of 2,600 µL.
- [ ] d. The Qubit indicates you to prepare volume for 13 samples to have a little more that is needed.

:::warning
:warning: Do not mix the working solution in a glass container.
:::

<p style="text-align: center;">Figure X - Qubit home screen: Activate the Reagent Calculator button (#1).</p>


<p style="text-align: center;">Figure X - Qubit Reagent Calculator.</p>


:::info
:info: Select Include overage: this allow the user to include an extra tube in the final volume calculation.
:::

- [ ] 4. Add the working solution to the microtubes.
* Standards
* - [ ] 190 µL of the working solution per microtube
* Samples
* - [ ] 199 µL of the working solution per microtube

- [ ] 5. Add the samples
* Standards
* 10 µL of the standard to their corresponding microtube and mix it by vortexing 2–3 seconds avoid creating bubbles.
* Samples
* 1 µL of the standard to their corresponding microtube and mix it by vortexing 2–3 seconds avoid creating bubbles.
- [ ] 6. Incubate the samples and standards for 2 minutes at room temperature.
- [ ] 7. Now select the assay type
* - [ ] dsDNA HS (High Sensitivity)

<p style="text-align: center;">Figure X - Qubit home screen: Choose an assay - dsDNA.</p>


<p style="text-align: center;">Figure X - Qubit choose an assay screen: dsDNA - High sesitivity.</p>


- [ ] 8. Read the standards

<p style="text-align: center;">Figure X - dsDNA: High sensitivity screen: Read the standards.</p>


<p style="text-align: center;">Figure X - dsDNA: High sensitivity screen: Read standard 1.</p>


a. Now read standard 2.

- [ ] 9. Now read the samples and start preparing the pooled sample.

#### Diaprepes samples

|Sample Number|[DNA] (ng/µL)|Volume (µL)|
|:-------------:|:-------------:|:-----------:|
|1|32.8|11|
|2|29.6|11|
|3|41.2|11|
|4|44.0|11|
|5|41.6|11|
|6|27.4|11|

#### Plant samples

|Sample Number|[DNA] (ng/µL)|Volume (µL)|
|:-------------:|:-------------:|:-----------:|
|1|29.0|11|
|2|41.8|11|
|3|28.2|11|
|4|34.8|11|

### Pooling the samples

#### Computing the volume to have equimolar amounts of each sample
* Steps
* - [ ] 1) Identify the sample with the highest [DNA].
* - [ ] a) Diaprepes: 44.0 ng/µL is the highest concentration from the Diaprepes samples.
* - [ ] b) Plant: 41.8 ng/µL is the highest concentration from the plant samples
* - [ ] 2) Divide that number between all the concentrations of the samples to determine the amount of µL to be tranfered to combine all the samples.

#### Diaprepes

|Sample No.|Calculation|Volume to be transferred (µL)|
|:----------:|:-----------:|:-----------------------------:|
|1|44.0/32.8|1.3|
|2|44.0/29.6|1.5|
|3|44.0/41.2|1.1|
|4|44.0/44.0|1.0|
|5|44.0/41.6|1.1|
|6|44.0/27.4|1.6|

#### Plant

|Sample No.|Calculation|Volume to be transferred (µL)|
|:----------:|:-----------:|:-----------------------------:|
|1|41.8/29.0|1.4|
|2|41.8/41.8|1.0|
|3|41.8/28.2|1.5|
|4|41.8/34.8|1.2|

*
* - [ ] 3) Transfer the computed volume for each sample to a clean microtube.
* - [ ] 4) Determine the total volume in the pooled microtube and multiply the volume the highest [DNA] determined in step 1.
* Diaprepes
* - [ ] Total Volume = 1.3 + 1.5 + 1.1 + 1.0 + 1.1 + 1.6 = **7.6 µL**
* - [ ] Total DNA ng = 7.6 µL x 44.0 ng/µL = **334.4 ng**
* Plant
* - [ ] Total Volume = 1.4 + 1.0 + 1.5 + 1.2 = **5.1 µL**
* - [ ] Total DNA ng = 5.1 µL x 41.8 ng/µL = **213.18 ng**
* - [ ] 5) Connect to [NEBioCalculator](https://nebiocalculator.neb.com/#!/dsdnaamt) to determine the fmoles of DNA in your pooled sample.
* - [ ] Diaprepes
* - [ ] Total DNA fmol = 360.8 fmol
* - [ ] Plant
* - [ ] Total DNA fmol = XXXXX fmol
* - [ ] 6) Determine the amount of volume needed to have 20 fmols for the next step in the protocol that is to add the adapters.
*