External Sequencing Preparation
Plasmid or Spanning PCR?
There are two distinct approaches to this, either sequencing the plasmid directly, or amplifying the F1-R2 insert region, and sequencing that. The advantage of sequencing the plasmid directly is that there is no chance of PCR mutations influencing the sequence, but the disadvantage is that we tend to get worse sequencing reads from plasmids compared to PCR products.
In either case, PURITY of the DNA is critical for the success of sequencing. A good strategy is to first attempt to sequence the plasmid directly. If that fails, then amplify the insert region and sequence that. Both products must be column purified before sequencing.
Sequencing Primers:
The usual primers to use for sequencing would be the F1-R2 primers in the diagram above.
However, for large inserts (>1 kb), you will need to design and order additional primers, since the average sequencing read length is only 500-700 bp, depending on template quality. In that case, space additional primers about 400 bp from the F1 and R2 primers, to ensure you get coverage of the entire insert sequence. (and for very large inserts, place further primers again ~400 bp away from the second set of primers).
For maximum confidence in the sequence, it is good practice to sequence BOTH STRANDS of the DNA, ie. to ensure you have duplicated sequence data all the way along the insert DNA. This will double your costs, but also your confidence.
Some companies require you to use primers that anneal at a specific temperature to facilitate their high throughput analysis. I’ve mostly seen this be 65°C.
Note that each sequencing reaction only contains ONE PRIMER! It is not a PCR! Therefore you’ll need to pay and prepare for twice as much sequencing if you want to sequence both the forward and reverse strands.
Preparation of Plasmid DNA for Sequencing
(Oh look, it’s just another miniprep - click for fully fleshed out guide)
Equipment and Consumables:
100 ml sterile LB Liquid Media in sterile culture flask
Oven or Heat Block at 60°C
250 μL Buffer P2 Lysis Buffer
500 μl Buffer PB Wash Buffer 1
750 μl Buffer PE Wash Buffer 2
30-50 μl Water Elution Buffer
Heat Block or Incubator at 37°C
Vortex
Protocol:
DAY 1: INOCULATION
Using the plasmid map as a guide add 100 µl of the appropriate antibiotic to your 100 ml of sterile LB media.
Honestly 5-15 ml of LB will also suffice. You can probably scale down this experiment without much trouble.
Set up a sterile workspace.
Flame your inoculating loop and use sterile technique to transfer the colony confirmed by the Colony PCRs to the flask.
Leave the lid loose enough for air transfer, then incubate at 37 degrees with shaking 8-16 hrs or overnight.
DAY 2: MINIPREP
Pellet cells in two 50 ml Falcon tubes by centrifuging at max speed for 5 minutes. You can balanace the centrifuge by adding sterile RO water to the lighter sample.
Pour off the supernatants.
Resuspend pelleted bacterial cells in 250 μl Buffer P1 Resuspension Buffer by vortexing or by using your micropipette to disturb the cell pellet. Transfer all of the suspension to a microcentrifuge tube.
Return the tube of P1 to the fridge as soon as you're done - it contains RNAse which will slowly denature at RT.
Equip yourself with a lab-coat and gloves if you haven't already and consider wearing goggles. The next few buffers range from mild to extremely toxic, consult the buffer mixing guides to learn more.
Add 250 μL Buffer P2 Lysis Buffer to each tube and invert gently 8-10 times. Incubate the tubes on the bench for 10 minutes.
Note; no vigorous shaking from here onward, you will shear your plasmid DNA.
Add 350 μL Buffer N3 Neutralisation Buffer to each tube and invert gently 8-10 times.
You should see a thick precipitate form, white if you are not using indicator. If you are using indicator dye, keep inverting until all traces of blue disappear.Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
Always be extremely careful to balance your centrifuge before spinning.
If your centrifuge can't go this fast the pellet will not be very compact, but you can overcome this with careful pipetting in the next step.
While you wait, assemble your silica spin columns and capture tubes. Label them carefully to match their corresponding tubes (currently spinning in the centrifuge). Also label a fresh set of 1.5 ml centrifuge tubes with the same label - these will be the tubes we eventually elute our DNA into for storage in the freezer.
Once the spin finishes, line all the tubes up in your tray to minimise the risk of pipetting errors.Carefully remove the supernatant without disturbing the white pellet of cell debris and decant it into the top of the silica spin column.
You can throw out the tubes containing the white precipitate, all your plasmid DNA is in the supernatant.
Centrifuge the silica spin columns in their capture tubes for 60 s at max speed.
If you notice some of the liquid hasn't passed through the silica then your max speed may be a bit low. Compensate by spinning for longer.
Optional: You can repeat this plasmid capture step step to improve yield. .Open the centrifuge and one-by-one remove the silica column + capture tube, pick up the flow-through in the capture tube with your pipette and add it back into the top of the spin column. Don't worry about it falling through the silica and onto the bench, it won't. Respin the tube 60s, max speed, to push the plasmid DNA through the silica filter a second time, potentially doubling the amount captured.
Discard the flow through into your miniprep waste container, it should not go with other liquid waste. Remember to follow proper disposal protocols.
Your plasmid DNA should now be bound to the silica column.
Wash the silica spin column by adding 500 μl Buffer PB Wash Buffer 1 and centrifuging for 60 s. Discard the flow-through into miniprep waste.
Wash the silica spin column by adding 750 μl Buffer PE Wash Buffer 2 and centrifuging for 60 s. Discard the flow-through into miniprep waste.
Centrifuge silica columns at full speed for an additional 1 min to remove residual wash buffer.
Remove silica columns from their capture tubes and place on a kimwipe and into 50°C Oven for 15-30 minutes
Alternatively place the columns into fresh capture tubes and leave in a heat block at 60°C for 15-30 minutes
Place the silica spin column in the clean 1.5 ml microcentrifuge tube that you labelled earlier. To elute DNA, add 30-50 μl Sterile RO Water to the center of each silica spin column, let stand for 1 min, and centrifuge at max speed for 1 min.
Water will pose the least risk of inhibiting the sequencing.
If you’re noticing significant volume loss at this stage, consider increasing the length or speed of the spin.
Optional: As with the capture spin, you can repeat this elution step with the same volume of liquid to potentially increase your yield. Using a micropipette, extract the eluted Plasmid DNA and place it back onto the spin column , let it stand for 1 min, then centrifuge at max speed for 1 min.
Dispose of the spin column, then fasten the lid on your microcentrifuge tube. Double check the label before storing at -20°C.
Send to external sequencing provider using the instructions on their website. Their requirements might be slightly different, but we prepare the following mixture;
Each mixture should contain:
Column-purified Plasmid DNA: typically 1-2 uL (10-75 ng)
Sequencing primer: 2 uL of 5 uM stock solution. When sequencing a PCR product, we usually usually one of the initial PCR primers as a sequencing primer too but note that the sequencing primer solution is 10x more dilute. Use sterile MQ to make the dilution. Note that a sequencing reaction only needs ONE primer, not two. It is not a PCR.
Sterile MilliQ water: Remainder of mixture, to total of 12 uL (or as specified by sequencing company)
Preparation of PCR Product for Sequencing
Thermal Cycler aka. Thermocycler aka. PCR Machine
This machine heats and cools in specific cycles.
Sterile Micropipette and tips
Sterile Workspace
Purified Plasmid DNA from above protocol
Fresh LB-Antibiotic plate (with antibiotic matching resistance gene on plasmid of interest)
100 ml Sterile LB Liquid Media
Inoculating Wand
Sterile PCR Tubes
Sterile Eppendorf Tubes
Sterile RO/MilliQ Water (dH2O)
.dna Map of the Region you plan to replicate, complete with information about primer binding, and length of amplicon. you should have your primer positions mapped out, matching the diagram above.
DNA Polymerase
Appropriate DNA Polymerase Buffer
Forward Primer F1
Reverse Primer R2
Additional forward or reverse primers if your insert is > 1kb to ensure maximum coverage (as explained above)
dNTPs
TE Buffer
PB Buffer
Silica capture columns
Note that not all columns are the same. You should ensure that you have silica spin columns that match the size of the PCR fragments you will amplify.
Protocol:
While you don’t technically need more than one PCR tube for this reaction, you may want to consider setting up a master mix for 4 tubes if your pipetting tolerance cannot handle these smaller volumes. Set up the following reaction on ice:
REACTION MIX (Final Conc.)
20 µl Sterile dH2O
2.5 µl 10x buffer (1 x)
0.5 µl 10 mM dNTPs (200 mM)
0.25 µl 50 µM Primer #1 (0.5 µM)
0.25 µl 50 µM Primer #2 (0.5 µM)
0.25 µl (5 U/µl) DNA Polymerase (0.05 U / µl)
1µl purified plasmid DNA in water
2. Set up the thermal cycler according to the following conditions;
THERMAL CYCLER SET-UP
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Initial denaturation: 95°C, 5 min
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Denaturation: 95°C, 30 sec, 25-35 cycles
Annealing: X°C, 30 sec, 25-35 cycles
Extension: 72°C, Y min, 25-35 cycles
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Final extension: 72°C, 10 min
Hold: 15°C
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The letters that are underlined and in bold indicate variables that need to be optimised for every individual PCR. Ideally you know these for your specific primers already, but I’ll include these notes just in case:
X = Annealing Temperature which primers will bind
Y = Extension Time that the polymerase will need in order to amplify your segment.
*OPPORTUNITY TO SLEEP WHILE THE THERMOCYCLER RUNS*
3. Combine your 4 PCR reactions (100 µl total) together in a microcentrifuge tube (OR) Pipette your 25 µl of PCR reaction to a microcentrifuge tube and make it up to 100 µl using TE buffer.
4. Put on all your PPE (Gloves, lab coat and goggles). Add 500 µl PB to your reaction mixture and invert to mix well.
5. Place an appropriate size-grade silica-based spin column into its 2 ml catch tube
6. Load 500 µl of the DNA-PB mixture onto the column. Spin at ~10,000 g for 30 sec. Discard the flow-through into culture waste.
7. Add 750 ml of buffer PE to the column, spin ~10,000 g for 30 sec, discard flow-through.
8. Repeat step 7.
9. Spin again for 30 sec to remove all traces of PE from the column. Discard both the flow-through and catch tube, and transfer the spin column onto a clean Kimwipe. Leave the column lid open. Transfer Kimwipe to 60°C oven, and allow to dry for 10 min.
10. Transfer spin column to a sterile 1.5 ml Eppi tube, and add 15 µl of Sterile RO Water to the centre of the spin column – ie on the membrane, not the walls of tube.
11. Allow to sit for 2 min. Spin at ~10,000 g for 1 min, retain Eppi tube with DNA solution in EB, discard spin column.
12. Set up the following (or similar according to their rules) reaction mixture to send to your external sequencing company. Each mixture should contain:
Column-purified Plasmid DNA: typically 1-2 uL (10-75 ng)
Sequencing primer: 2 uL of 5 uM stock solution. When sequencing a PCR product, we usually usually one of the initial PCR primers as a sequencing primer too but note that the sequencing primer solution is 10x more dilute. Use sterile MQ to make the dilution. Note that a sequencing reaction only needs ONE primer, not two. It is not a PCR.
Sterile MilliQ water: Remainder of mixture, to total of 12 uL (or as specified by sequencing company)
Acknowledgements:
Coleman Protocols 2017 + 2019 http://coleman-lab.org/