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# System prepended metadata

title: Protocols

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#### Protocols
##### Media
LB media was made using LB Broth (Lennox) from Sigma-Aldrich. 1 L broth was prepared by dissolving 20 g of LB Broth (Lennox) in 1 L of distilled water, and thereafter autoclaving the mixture. The finished media was kept at room temperature.

LB media with agar was using LB Broth with agar (Miller) from Sigma-Aldrich. 1 L was prepared by dissolving 40 g of LB Broth with agar (Miller) in 1 L of distilled water. The mixture was then autoclaved and put into a 60°C cabinet.

For liquid media or plates supplemented with antibiotics, 1000x dilutions were added to the media. This gave final concentrations of ampicillin (100 &mu;g/mL), chloramphenicol (34 &mu;g/mL), tetracycline (10 &mu;g/mL), and kanamycin (50 &mu;g/mL). 

##### Chemical competent cells 
Adaption of protocols provided by R. Palm and J. Mejlsted.

A 3 day protocol for generating roughly 80x 50 ul aliquots for transformation protocols.

Materials:
Buffers
* 0.1 M CaCl2 (5.55 g in 500 mL)
* 0.1 M CaCl2/10% glycerol (in 50 mL 0.1 M CaCl2 add 500 uL glycerol 100%)
Media
* LB media
* LB agar plate
Other components
* An aliquot of competent E. coli cells
* Sterile 0.5 mL microtubes
* Sterile Erlenmeyer flasks
* Falcon tubes

Procedure:
Day 1
1. Around 4PM: Plate roughly 20 uL of competent cells on a LB agar plate without antibiotics.
2. Incubate plate at 37°C between 16-20 hours.

Day 2
3. At around 10 AM: Pick a single colony from the plate, and let it grow in 100 mL LB media at 37°C and 200 rpm for 6 to 8 hours.
4. At around 6 PM: Add 250 mL LB to a sterile Erlenmeyer flask. Add around 25 uL to 1 mL of culture to the media.
5. Let the culture grow overnight at 37°C.
6. Place buffers in the fridge.
7. Place pipettes, falcon tubes, and sterile 0.5 mL microtubes into the freezer.

Day 3
8. Measure OD600 of the overnight culture, and inoculate a Erlenmeyer flask with a volume so the final OD600 value in the culture becomes 0.01.
9. Grow the culture at 37°C with shaking, and measure OD regularly.
10. When an OD between 0.3-0.55 is reached, split up culture into 10x 50 mL falcon tubes (Each of them will have 25 mL of culture).

**From here on it is important that the cells, buffer and equipment remain at low temperature.**

11. Centrifuge the cells at 4000 rcf for 10 min at 4°C.
12. Add to each falcon tube 10 mL ice-cold 0.1 M CaCl2 and resuspend pellet by shaking the Falcon tubes (if possible avoid vortexing and repipetting).
13. Centrifuge the cells at 4000 rcf for 10 min at 4°C.
14. Resuspend pellet in 400 uL 0.1 M CaCl2/10%glycerol (vortex to resuspend the pellet).
15. Dispense 50 uL aliquots of suspension into 0.5 mL microtubes.
16. Store competent E. coli cells in -80°C.

##### X7 PCR
Materials:
* rCutSmart buffer
* dNTPs (2 mM)
* Sterile MQ
* Primers (10 pmol/uL)
* X7 polymerase (2-3 U)
* DNA template (plasmid: 10 pg/uL)
* PCR tubes
* Thermocycler
* DMSO (optional)

Procedure:
1. Mix following in PCR tubes (adjust “# of reactions” if needed):
<!DOCTYPE html>
<html>
<head>
<style>
table {
  font-family: arial, sans-serif;
  border-collapse: collapse;
  width: 100%;
}

td, th {
  border: 1px solid #dddddd;
  text-align: left;
  padding: 8px;
}

tr:nth-child(even) {
  background-color: #dddddd;
}
</style>
</head>
<body>

<table>
  <tr>
    <th>Reagent</th>
    <th>Volume per reaction (uL)</th>
    <th>Mastermix per volume (uL)</th>
  </tr>
  <tr>
    <td>rCutsmart</td>
    <td>10</td>
    <td>20</td>
  </tr>
  <tr>
    <td>dNTPs (2 mM)</td>
    <td>5</td>
    <td>10</td>
  </tr>
  <tr>
    <td>MQ</td>
    <td>31.5</td>
    <td>63</td>
  </tr>
  <tr>
    <td>Primer 1 (10 pmol/uL)</td>
    <td>1</td>
    <td>2</td>
  </tr>
  <tr>
    <td>Primer 2 (10 pmol/uL)</td>
    <td>1</td>
    <td>2</td>
  </tr>
  <tr>
    <td>X7 enzyme</td>
    <td>0.5</td>
    <td>1</td>
  </tr>
   <tr>
    <td>Template</td>
    <td>1</td>
    <td>-</td>
  </tr>
   <tr>
    <td></td>
    <td># of reactions</td>
    <td>Total volume</td>
  </tr>
  tr>
    <td></td>
    <td>2</td>
    <td>98</td>
  </tr>
</table>

</body>
</html>

2. Run the following program in a thermocycler (adjust elongation time to fragment size):
<!DOCTYPE html>
<html>
<head>
<style>
table {
  font-family: arial, sans-serif;
  border-collapse: collapse;
  width: 100%;
}

td, th {
  border: 1px solid #dddddd;
  text-align: left;
  padding: 8px;
}

tr:nth-child(even) {
  background-color: #dddddd;
}
</style>
</head>
<body>


<table>
  <tr>
    <th>Step</th>
    <th>Temperature (°C)</th>
    <th>Time</th>
    <th>Cycles</th>
  </tr>
  <tr>
    <td>Activation</td>
    <td>98</td>
    <td>30 s</td>
    <td></td>
  </tr>
  <tr>
    <td>Denaturation</td>
    <td>98</td>
    <td>10 s</td>
    <td>x35</td>
  </tr>
  <tr>
    <td>Annealing</td>
    <td>68 -0.5/cycles</td>
    <td>30 s</td>
    <td>x35</td>
  </tr>
  <tr>
    <td>Elongation</td>
    <td>72</td>
    <td>1 min/kb</td>
    <td>x35</td>
  </tr>
  <tr>
    <td>End elongation</td>
    <td>72</td>
    <td>10 min</td>
    <td></td>
  </tr>
   <tr>
    <td>Hold</td>
    <td>12</td>
    <td>Infinite</td>
    <td></td>
  </tr>
</table>

</body>
</html>

3. DpnI digestion of the template backbone (**Not needed if you do gel-band purification**):
    Add 1 µL DpnI to the clean PCR product.
    Incubate 30 min at 37°C (or 1 hr if the template is gDNA).
    Heat inactivate 20 min at 80°C.
4. Run a gel of 3 µL PCR product with dye to check for the correct band lengths.
    If there is only the correct band: PCR clean up.
    If there are multiple bands: run gel with remaining PCR product, excise, and gel purify.
5. Measure concentration of the fresh fragment using a NanoDrop.


##### Plasmid transformation of *E. coli*
According to protocol by GoldBio (https://goldbio.com/documents/4066/DH5-alpha%20Chemically%20Competent%20E.%20coli%20cells%20Transformation%20Protocol.pdf).

Materials:
* Competent E. coli
* LB with appropriate antibiotics
* 1-5 mL tubes
* Ice
* SOC media (recovery media, optional), otherwise LB
* Thermoshaker

Procedure:
1. Remove competent cells from -80°C freezer and thaw completely on ice (10-15 min).
2. Aliquot 1-5 µl (1 pg-100 ng) of DNA to the chilled microcentrifuge tubes on ice.
3. When the cells are thawed, add 50 µl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. Mix well by tapping. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency.
4. Incubate the cells with DNA on ice for 15 minutes. (BL21: 30 min).
5. After the 15-minute ice incubation, heat shock the cells at 42°C for 45 seconds. (BL21: 10 s).
6. Transfer the tubes to ice for 2 minutes. (BL21: 5 min).
7. SOC media (optional, not needed for ampicillin marker).
    Add 950 µl of Recovery Medium or any other medium of choice to each tube.
    Incubate tubes at 37°C for 1 hour at 210 rpm in a shaker incubator.
8. Spread 50 µl to 200 µl from each transformation on prewarmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies.
9. Incubate the plates overnight at 37°C.


##### USER cloning
Materials:
* BioBLock DNA with USER overhangs
* USER-compatible opened vector
* CutSmart buffer
* Sterile MQ
* USER Enzyme Mix
* Competent *E. coli* (DH5&alpha;)
* Ice
* LB plates supplemented with appropriate antibiotics
* PCR tubes

Before conducting the cloning:
    Keep total volume of mix at 10 &mu;L (or alternatively 20 &mu;L).
Add equimolar amounts of each fragment.
    

Procedure:
USER reaction mix
1. Mix following in a PCR tube:

<!DOCTYPE html>
<html>
<head>
<style>
table {
  font-family: arial, sans-serif;
  border-collapse: collapse;
  width: 100%;
}

td, th {
  border: 1px solid #dddddd;
  text-align: left;
  padding: 8px;
}

tr:nth-child(even) {
  background-color: #dddddd;
}
</style>
</head>
<body>


<table>
<tr>
   	<th>Reagent</th>
    <th>Volume (&mu;L)</th>
    <th>Negative control (&mu;L)</th>
  </tr>
  <tr>
   	<td>BioBlock DNA</td>
    <td>1-6</td>
    <td>0</td>
  </tr>
  <tr>
    <td>USER-ready vector</td>
    <td>1-6</td>
    <td>1-6</td>
  </tr>
  <tr>
    <td>CutSmart buffer</td>
    <td>0.5</td>
    <td>0.5</td>
  </tr>
  <tr>
    <td>MQ (up to 10)</td>
    <td>x</td>
    <td>x</td>
  </tr>
  <tr>
    <td>USER Enzyme</td>
    <td>1</td>
    <td>1</td>
  </tr>
</table>

</body>
</html>

Incubate as follows:
2. 37°C for 25-35 min.
3. Room temperature for 15-25 min.
4. Thaw competent *E. coli* DH5&alpha; on ice, while they are thawing:
5. Continue incubation for app. 10 min.
6. Put on ice, do not mix, do not vortex.

Transformation:
7. Mix the reaction with 10-50 &mu;L competent *E. coli* DH5&alpha; - Competent cells should be handled gently.
8. Mix the DNA and cells gently by stirring with a pipette tip.
    Remember to add a positive control, using an unopened plasmid (typically 1 pg - 100 ng).
9. Incubate on ice for 5-15 min
10. Heat shock at 42°C for 0:50 - 1:30 min
11. Incubate on ice for 5-15 min
12. (Not needed for ampicillin marker) Add 950 µl SOC/LB media and incubate for 1 h at 37°C 250 rpm)
13. Plate and spread on selective medium (LB + Amp, Kan/Neo or Cam)
    

##### Invitrogen(TM) PureLink(TM) Quick Plasmid Miniprep Kit
https://assets.fishersci.com/TFS-Assets/LSG/manuals/purelink_quick_plasmid_qrc.pdf
Miniprep isolation protocol (centrifuge)

Follow this procedure to purify plasmid DNA using a centrifuge. Use a microcentrifuge capable of
centrifuging at >12,000 × g. For processing a large number of samples simultaneously, see the "Miniprep
plasmid isolation protocol (vacuum)".

* Perform all centrifugation steps at room temperature using a microcentrifuge.
* Optional: Preheat an aliquot of TE Buffer (TE) to 65–70°C for eluting DNA. Heating is optional for eluting 1–30 kb plasmid DNA but is recommended for eluting DNA >30 kb.
* Ensure the bag containing the PureLink™ Quick Spin Columns is closed tightly after each use.
* Caution: Buffers contain hazardous reagents. Use caution when handling buffers.

Steps:
1. Harvest
    Centrifuge 1–5 mL of the overnight LB-culture. (Use 1–2 × 10^9 E. coli cells for each sample.) Remove all medium.
2. Resuspend
    Add 250 &mu;L Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is     homogeneous.
3. Lyse
    Add 250 &mu;L Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes.
4. Precipitate
    Add 350 &mu;L Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 10 minutes. 
5. Bind
    Load the supernatant from step 4 onto a spin column in a 2 mL wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the flowthrough and place the column back into the wash tube.
6. Wash (Optional)
    (Recommended for endA+ strains). Add 500 &mu;L Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Centrifuge the column at 12,000 × g for 1 minute. Discard the flowthrough and place column back into the wash tube.
7. Wash and remove ethanol
    Add 700 &mu;L Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 × g for 1 minute. Discard the flowthrough and place the column into the wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the wash tube with the flowthrough. 
8. Elute
    Place the Spin Column in a clean 1.5 mL elution tube. Add 75 &mu;L of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature. 
9. Recover
    Centrifuge the column at 12,000 × g for 2 minutes. The elution tube contains the purified plasmid DNA. Discard the column. Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C (long term).


##### Monarch® Plasmid Miniprep Kit from NEB
https://www.neb.com/en/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).
If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve.
Store Plasmid Neutralization Buffer (B3) at 4°C after opening, as it contains RNase A.

Note: unlike other commercial kits, all wash steps are required.

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.

*Note: For a standard miniprep to prepare DNA for restriction digestion or PCR, we recommend 1.5 ml of culture, as this is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).*

2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1)  (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2)  (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.

*Note: Care should be taken not to handle the sample roughly and risk shearing chromosomal DNA, which will co-purify as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.*

4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3)  (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.

*Note: Be careful not to shear chromosomal DNA by vortexing or vigorous shaking. Firmly inverting the tube promotes good mixing, important for full neutralization.*

5. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.

*Note: Spin time should not be less than 2 minutes. Careful handling of the tube will ensure no debris is transferred and the 2 minute recommended spin can be successfully employed to save valuable time. For culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.*

  *To save time, spin for two minutes only.*

6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through. 

*To save time, spin for 30 seconds, instead of 1 minute.*

 *If using a vacuum manifold instead of centrifugation, insert the column into a manifold and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.*

7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.

*Note: The collection tube is designed to hold 800 &mu;L of flow-through fluid and still allow the tip of the column to be safely above the top of the liquid. Empty the tube whenever necessary to ensure the column tip and flow-through do not make contact.*

 *To save time, spin for 30 seconds, instead of 1 minute.*

*If using a vacuum manifold, add 200 &mu;L of Plasmid Wash Buffer 1 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.*
*Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.*

8. Add 400 &mu;L of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

  *When using a manifold add 400 &mu;L of Plasmid Wash Buffer 2 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.*

9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.

  *If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt and ethanol are carried over to the next step.*

10. Add ≥ 30 &mu;L DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

*Note: Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Delivery of the Monarch DNA Elution Buffer should be made directly to the center of the column to ensure the matrix is completely covered for maximal efficiency of elution. Additionally, yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated as a result of dilution. For larger plasmids (≥ 10 kb), heating the DNA Elution Buffer to 50°C prior to eluting and extending the incubation time after buffer addition to 5 minutes can improve yield.*
 

##### Protocol for Ampliqon PureIT ExoZAP PCR CleanUp 
https://ampliqon.com/en/pcr-enzymes/pcr-enzymes/pcr-clean-up/pureit-exozap-pcr-cleanup/

This protocol serves as a guideline for clean-up of 5 μl PCR product using PureIT ExoZAP PCR CleanUp*.
Take out PureIT ExoZAP PCR CleanUp from the -20 °C freezer.

* Keep PureIT ExoZAP PCR CleanUp on ice at all times.
* Add 2 μl PureIT ExoZAP PCR CleanUp** to 5 μl of amplified PCR product.
* Mix well and spin down.
* Incubate the reaction at 37 °C for 2-5 minutes to degrade remaining primers, single-stranded DNA and to inactivate excess nucleotides by dephosphorylation.
* Incubate at 80 °C to completely inactivate PureIT ExoZAP PCR CleanUp for 3-10 minutes.
* The cleaned up PCR product can now be used for downstream applications such DNA sequencing, primer extension experiment or SNP analysis.
* After treatment the PCR products can be stored at -20 °C

*If treating PCR product of higher volume, then increase proportionally the amount of PureIT ExoZAP PCR CleanUp.
**PureIT ExoZAP PCR CleanUp works in PCR buffers.



