Cloning and Sequencing woes - (Feb/05/2008 )

I have been trying to sequence several DNA libraries without much success for months. Here's the summary of what had been done:

1. A library of 154 bp templates was amplified by Taq polymerase, gel purified, endpolished (using T4 DNA polymerase and PNK) and ligated into a blunt vector (EcoRV cut, pZERO1 vector).

Ligation ratio: 33 ng of pZERO1 vector (2.8 kb) and 20-50 ng 154-bp PCR product.
Ligation conditions: In Invitrogen's ligase buffer, 5 U T4 ligase, overnight incubation at 16oC

After ligation, the reaction was purified by phenol chloroform extraction and resuspended in 20 ul h20.

2. 1 ul of the purified ligation reaction was electrotransformed into TOP10 oneshot cells, and plated onto Zeocin plates. (The selection is by positive selection, ie. only recombinants are expected to grow.)

3. After ON incubation at 37oC, colony PCR was done on 48 random colonies. At least 95% of colonies bore at least one copy of the 154 bp insert.

4. The same transformation mixture was then plated onto large agar plates for robotic picking.
After ON incubation in liquid broth, Templiphi reaction was done to amplify the plasmid.

5. Sequencing was done the templiphi reaction. A Templiphi control using another plasmid template was done.

6. RESULTS: No sequence results.

Troubleshooting
1. Templiphi control reaction managed to sequence.
2. Colony PCR was done on the Templiphi reaction, and the recombinant plasmid template was present
1& 2 indicate that the culture did grow, and the templiphi reaction was fine.

WHAT COULD BE THE PROBLEM HERE?
Someone suggested that it is more difficult to sequence when concatemers are ligated into the the vector.
Could the cloning strategy be improved?
-I tried to end polish the purified PCR product with just T4 DNA pol, and use the ZERO Blunt cloning kit, but no colonies grew on the Zeocin plates.


Appreciate if someone can spend time to read through this and offer suggestions. If I am unclear, please feel free to ask.

Thanks a lot!

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How was the templiphi reaction primed? Did you use random hexamers or plasmid-specific primers? The primer binding site could be missing if you used specific primers. How was the sequencing reaction primed? Could your sequencing primer be the problem?

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The Templiphi was primed with random hexamers. I managed to amplified the region containing my insert so the templiphi worked and generated the template.

Am in the midst of troubleshooting whether the sequencing primer was the problem. It's frustrating because I have had success with one library and couldn't never seem to replicate it anymore.

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If you are able to do PCR with some primers, you should also be able to sequence with those same primers. This would suggest (1) try both of those primers (2) the problem is with purity or amounts of DNA. How are you purifying the DNA for sequencing? How much are you using? Too much or too little will both cause problems.

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silly question here, if the Templiphi reaction isn't working too well, why not just skip it and sequence your plasmid with an M13 primer? If you have the plasmid, you can directly use the plasmid as a template for a sequencing reaction. I don't see a need to conduct a Templiphi reaction.

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Templiphi allows you to use dramatically smaller amounts of plasmid for sequencing. This is great if you have low copy plasmids or limited amounts of template.
A typical sequencing reaction needs quite a bit of DNA relative to other things such as transformation or PCR.

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I see. Thanks

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Additional notes:

1. The templiphi gave high molecular weight product, so it went well.

2. I am now wondering whether chimeras formed during the Templiphi reaction might have interfered with the sequencing. For my one and only single set of "acceptable" and "analyzeable" sequencing results from a plate of 384 templiphi products, I noticed there were unexpected sequences in the insert, especially when the clone contains concatemers of the insert. (My insert carries fixed adaptor sequences.)

Unfortunately since then, the in-house cloning and sequencing facility, has decided to pick just 10 samples from 384-well templiphi plate to check that the sequencing passes (>70% pass rate) before sequencing the rest of the plate, so I can't even get 5 decent sequences (either low or no signal in sequencing) to do any preliminary analysis.

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As far as I am aware, every sequencing center using capillary sequencing has switched to templihi amplification of plasmids, so if there were a consistent problem such as this, I think we would know about it.

Tell us more about the process... where does the DNA come from initially, how is it purified, how are the reactions run. You may be amplifying lots of DNA which is not your desired sequencing template.

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For the direct sequencing here's what happened, right from the beginning.
- PCR product was blunt-ligated into an EcoRV cut pZERO vector, electrotransformed into TOP10 cells, and plated onto small Zeocin agar plate.
- Colony PCR was done. Almost all colonies carry the insert of 1 (most common),2, and 3 copies....***I do see some colonies with multiple bands, and am not sure if multiple, different plasmids got in a single E coli cell.****
- Same transformation mix was used to plate onto large Q trays. Incubation ON.
Subsequently, the cloning and sequencing facility processed the plates as follows:
- Colonies picked by robot and inoculated into 384-well culture plate. Incubation ON.
- Templiphi on a small aliquot of the liquid culture. a pUC plasmid was included as control for Templiphi. Incubation ON.
- 10 Templiphi reactions from each 384-well plate were used for sequencing. Sequencing primer is one of the two M13 primers.
- Results: generally no signal or low signal.

The insert has this structure:
Adaptor1-Tag1-Adaptor2-Adaptor3-Adaptor4-Tag2-Adaptor5 (154 bp)
Tags are parts of human genome, and adaptors are non-human sequences.


As of now, I have managed to sequence the PCR-amplified Templiphi product successfully. Here's what I did:
-Deliberately selected those Templiphi wells that contain only one copy of my insert (to rule out the possibility that concatemers may be harder to sequence)
-PCR using M13 primers
-Purified the product
-Sequenced using the same conditions that failed in the direct sequencing of Templiphi. Sequencing primer is one of the two M13 primers.
-Results: OK.

Templiphi did seem to work because:
- the pUC templiphi control managed to sequence
- large molecular weight size products were seen when the templiphi products were ran on the gel.

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