single cell PCR

[badguy]

I'm trying to do Hot Start PCR using single buccal cell as my sample. I'm planning to put the cell directly into the PCR reagent mix.
Does this step workable? Recommended initial activation time is 95C 5min for Qiagen's HotStar PCR kit, should I increase the time to 10-15min for the initial denaturation?

[relaxin]

Are you doing genomic DNA PCR or RT-PCR? For genomic PCR, adding the cell directly to the PCR mix should work. Initial denaturation for 5 min should be OK. Longer denaturation may shorten the life of the polymerase.

If you are doing qRT-PCR, try use Ambion® Single Cell-to-CT™ Kit.

[mchlbrmn]

If it's single cell genomic, that means you're aiming to amplify only one or two copies of the target gene, which is challenging.

[badguy]

I'm gonna do single cell genomic PCR. I'm doing nested PCR for this.
5min denaturation sufficient to break the cells? Previously I set 15min denaturation time for my colony PCR.

[CrowSan]

Is the single cell mamalian or bacterial? The use of the word colony makes me suspect that it is the latter.

[mchlbrmn]

Oh, oops, you're right. It says "buccal" also, so I guess genomic bacterial should be somewhat easier than PCRing from genomic mammalian, and I should have said one instead of two copies of target. I don't know how challenging PCRing from a single unpurified bacterial cell is.

[relaxin]

I'm gonna do single cell genomic PCR. I'm doing nested PCR for this.
5min denaturation sufficient to break the cells? Previously I set 15min denaturation time for my colony PCR.

I do my colony PCR with only 5 min initial denaturation. So 5 min denaturation will be fine with buccal cells.

Guys, stop guessing. It is buccal cell, not bacterial cell. Badguy just mentioned previous experience with colony PCR.

[badguy]

I have experience on colony PCR.
Now I'm trying to do PCR with single buccal cell. So, it's a mammalian cell
So, don't confuse yourself.

[mchlbrmn]

I was confused, sorry.

[CrowSan]

Mia Culpa

[badguy]

I ended up getting smearing after electrophorosis.
man this is difficult.
Will small amount of PBS inhibit PCR reaction? I make the cell suspension in 1X PBS.

[relaxin]

Small volume of PBS should not interfere with PCR. What is the volume relative to that of the PCR mix?
Are you sure that you got a single cell or just PBS in the PCR mix?

[badguy]

the transfered volume of PBS is less or equal to 1ul. the exact amount is unknown because the cell is transfered using pasteur pipette.
Nothing was found in the pipette after the transfer.

[badguy]

I tried to run nested pcr using single cell. I'm using Qiagen's HotStar kit.
I use 1X PBS to do the cell suspension.

1st master mix as below:
5X Buffer - 4ul
5X Q-solution -4ul
7 primers (10uM)at final concentration of 0.2uM
Taq - 0.8(2 unit)
dh2o - top up to 20ul


2nd reaction master mix as below:
5X Buffer - 4ul
5X Q-solution -4ul
7 primers at final concentration of 0.2uM
Taq - 0.4(1 unit)
dh2o - top up to 20ul

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I ended up with smearing after the reaction. Could it be primer design is the problem?

[relaxin]

What are the 7 primers? Did the primer work in regular PCR? Do you have primers for a housekeeping gene?

What is the number of cycles did you use in the two PCRs?

Did you use nested primers for the second PCR?