Resuspend the cells(any number), smaller numbers also work, in 10-20 ul of TE buffer or milli Q water. boil for 5-10 min in a waterbath, quick chill, short spin. use the supernatant as a template for PCR.

If cells are very less, u can directly add cells to the PCR reaction without enzyme,boil for 5-10 min in a waterbath, let it cool slowly, short spin. add enzyme to the supernatant and start the reaction at extension step followed by normal PCR reaction steps.

Try this protocol, we used it to quantify mycobacteria in macrophages and then we estimated bacterial numbers per cell.

http://www.ncbi.nlm.nih.gov/pubmed/22532835

Thank you very much

Quanta sells a nonezymatic extraction kit that works on tissue cells and bliod

Dear Yeo

I would like to suggest you to go for DNA extraction before PCR reaction. I have tried the method suggested by Dr Suruchi (this protocol have been reported in many papers published in good quality journals) for genomic DNA extraction of bacteria but the result is not quite satisfactory. In my case the same sample showed negative result in one PCR reaction followed by positive result in the second reaction. Therefore I switch over to extraction of genomic DNA followed by PCR and got uniform result.

Dear Dr. Laxmi,

With all due respect, this perfectly works in our lab for mammalian cells. We have amplified the genes using normal cheek cells as well.

Dear all,

Just for sharing.

I had tried the method suggested by Dr. Suruchi and I did some modification and the condition worked perfectly fine. Briefly, after boiled the sample(in TE), I added some proteinase K and incubated at 37C for 1 h then boiled the sample again. It worked pretty well.

Hi Kok, I am currently having HeLa cell with transfected plasmid. I would like to do direct PCR on mammalian cells to verify my target plasmid. Do you mind to let me know how much proteinase K did you add into your boiled cells in TE?

@Pei Sheng :50ug proteinase K in 150uL reaction volume.

We did some tests as well based on what was described above, although we didn't push it to the limit.

Here is what we did:

* HeLa cells, 1/6th of a confluent well in 96-well format.

* Trypsinise cells & take aliquot in small volume (e.g. 50ul). Pellet cells (1min full speed in a table top should suffice) and remove supernatant.

* Wash by resuspending in 5-10mM Tris pH8.5, pellet again.

* Resuspend cells in 10ul of the above Tris buffer, heat 15' at 99 degrees Celcius.

* Cool on ice, centrifuge 1min at full speed.

* Use the entire supernatant directly in a PCR.

- We have tried a PCR (reaction of 50ul total) with Q5 polymerase (NEB), but I suppose that e.g. Taq should also work. Conditions may differ for each PCR product, therefore first optimise your reaction on purified DNA and don't forget to take along controls: purified DNA as a positive control and water-only as negative control.

- We didn't try less input material, but bands are very fat. So, you could probably scale down even further.

- Personally, I would not useTE, as EDTA might inhibit subsequent enzymatic (PCR) reactions.

- As mentioned above, ProtK may be needed if you use (much) more material.

 Thank you very much, Kok and Jeroen. I will try this method. Hopefully it works and I won't be rely on kit anymore. 

Hello Kok Siong Yeo

I want to ask what PCR product size you have tried with this protocol. Are there restrictions on how long you can amplify based on this quick boil protocol?

Thank you.

Jeroen, I have a question regarding your protocol. How did you set up your PCR afterwards? 25 ul or 50 ul?

 @Jenny

We used a 50ul final PCR reaction volume. I added this information now to the original answer.

However, as indicated there as well, we had very fat bands and I think that downscaling is possible. This might depend on your particular reaction though.

 Hi Donghui Li,

Sorry for the late reply.

We tested about 400-500bp only, we are not so sure how long it can go. 

Greetings!  Just take the OmniKlentaq or OmniTaq enzyme (www.klentaq.com). These are Taq mutants, specially designed for direct PCR w/o any extraction, due to their high resistance to inhibitors. Mammalian cells / lysates in particular are even not so challenging for them, and the direct PCR works like a charm.                                                 More details:  just call 314-771-5566.

Jeroen Strating, I've attempted your methodology with MDA-MB-231 cells from a fully confluent 6-well plate while scaling according to the confluency you used. Unfortunately, it did not work, though using trypsin with EDTA may be a potential problem.

Do you suggest pelleting straight from trypsinized solution or do you neutralize with complete growth media before pelleting? We'll try again with trypsin missing EDTA.

I encourage you to message me.

Kevin Farquhar

@Kevin Scott Farquhar

Usually we do not neutralise the trypsin. So, directly pellet the cells after trypsinisation (we use regular trypsin with EDTA). We do wash once in Tris buffer (see my original post) to remove the trypsin and EDTA (or at least most of it).

You wrote you use a fully confluent 6-well. There is a good chance you are completely overloading it. We use this protocol only with a very small amount of cells (like less than a confluent 96-well). Still, we use this protocol only with HeLa cells (never tested other cell types), so I have no clue on how your cells will behave.

One final question, just because you did not state it explicitly: Did you take a control for the PCR reaction (e.g. with plasmid DNA or highly purified genomic DNA as template)?

I hope you can get it to work.

Jeroen

Hello,

Just use the inhibition-resistant mutants of Taq, Omni Klentaq or OmniTaq, available from DNA Polymerase Technology (www.klentaq.com). Simply suspend the cells in 1X PCR buffer and proceed with PCR.  It works like a charm.

Greetings,

Milko

@Jeroen Strating

Thanks for the quick response.

We had a positive control reaction with genomic DNA as template, and it worked. I think a better positive control would be to lyse the clonal cells that the genomic DNA was purified from, since we know it should work. 

Also, when you washed the cells, you send you resuspended them (before boiling). How much Tris-HCl did you wash/resuspend the pellet with?

I'm going to modify the protocol as you described and see how it works. On

the follow protocols have been tested in our lab and all give rise to high yield with high purity.

1)  http://www.stembook.org/node/1438.html

2)RamleeMK 2015. High-throughput genotyping

of CRISPR/Cas9-mediated mutants

using fluorescent PCR-capillary gel

electrophoresis. Scientific reports

I personally find the 1) protocol works best.

Hope it helps.

Hi, you can use the PGD protocol.

Jeroen Strating Hi! Thank you so much for the protocol!

I've just tested it out on my lung cancer cell lines yesterday and the PCR looks perfect!

Thank you again!

Jeroen Strating, Thanks for the protocol. Suruchi Aggarwal - what temp is the water bath?