Diatom PCR


PAW-PCR Colony Screen
(October 2011 Dylan Scott)

Introduction

This protocol is designed to prepare marine diatom cells grown in liquid media for a PCR colony screen using gene specific primers. It is designed to mimic colony screens used to screen bacteria colonies when cloning. Because residual salt water from culture media can prevent successful PCR reactions, it is essential to remove all salt water from the cell sample before adding it to a PCR tube. To do this, we pellet, aspirate and wash (PAW) the diatom cells to remove all excess salt from the cell sample.
Diatom cultures that are to be screened should be “younger and happily growing” for best results. 10 days post culture inoculation (depending on culture and growth conditions) is a good aim for screening.
If growing diatom colonies on agar plates, plucked colonies can be added directly to PCR reactions (like a traditional bacteria colony screen). Plated colonies may be diluted in dH2O (see step 5).

PAW Preparation of Marine Diatom Cells

All steps should be promptly followed to prevent cell lysis when cells are resuspended in dH2O.
1.     Transfer 1.0 mL of diatom cells in culture media to a clean 1.5 mL tube and spin at max (13-16,000g) for 10 min in a microcentrifuge at room temperature.
a.     Since only a small sample of cells is needed for each PCR reaction, the starting concentration of cells is not overly important. Cells should be visible and well mixed before pipetting a sample to a 1.5 mL tube.
2.     Aspirate the supernatant. It is essential to remove all media from the pellet.
a.     Resuspend the pellet in 10µL PCR quality dH2O and transfer the pellet to a clean tube.
b.     Add 1.0 mL PCR quality dH2O and gently resuspend the transferred pellet.
3.     Centrifuge samples again at max for 10 min.
4.     Aspirate the supernatant.
5.     Resuspend the pellet in a minimal volume (10µL) of dH2O for addition to PCR reactions. Depending on the yield of final pellet (due to losses in transfers and resuspending the pellet), the cell sample can be diluted further.
·      There appears to be an inverse correlation between added cell sample and PCR yield. Remember, you could hypothetically get successful amplifcation from a single cell.
·      You can choose to serially dilute the final cell yield and run several different concentration of cells (figure 1; this is recommended for the first trial). I usually have a sufficient pellet to initially resuspend it in 10µL dH2O (so-called 1:1 dilution) before diluting it down to 1:1000.
6.     Add the washed, resuspended diatom pellet samples to individual PCR tubes with master mix. See the below table for recommended PCR mix.

Colony Screen PCR Mix

Reagent
1x
MM ( 16 x)
Primer 1 – Forward
0.5µL
  8.0  µL [√ ]
Primer 2 – Reverse
0.5µL
  8.0  µL [√ ]
dNTP
0.2µL
  3.2 µL  [√ ]
10x PCR Buffer*
1.0µL
 16.0 µL [√ ]
5x Q-solution**
2.0µL
 32.0 µL [√ ]
dH2O
3.75µL
 60.0 µL [√ ]
Taq polymerase
0.05µL
   0.8 µL [√ ]
subtotal
8.0µL
  128 µL [√ ]
Purified cell sample (DNA)
2.0µL

GRAND TOTAL
10µL

MM is master mix. Standard mix is in plain text. Examples are in italics.

* I use PCR Buffer with CoralLoad (Qiagen); CoralLoad PCR Buffer (containing 2 gel-tracking dyes) enables immediate loading of PCR product onto a gel.
** Q-solution (Qiagen) is optional. If excluded, adjust the final volume with dH2O.

8.0µL master mix added to each PCR reaction tube, before adding 2.0µL cell sample.

PCR Cycle settings:

94°C x 15 min [initial cell denature stage]
(94°C x 30s, *57°C x 30s, 72°C x 60s) x 35 cycles
72°C x 10 min [final extension]
*adjust the annealing temperature (57°C) to fit the Tm of your own primers (approximately 5°C below Tm)


Figure 1 – A serial dilution of diatom cell pellet, after initially resupending the pellet in 10µL dH2O. 2µL of each dilution was added to the PCR reaction.
 

1 comment:

  1. Hello Dylan, I'm working now with genetic engineering of microalgae, with Chlorella and Scenedesmus, I found quite useful your protocol and strategies, so I wanted to ask you something, After I performed the colony PCR from my transfected microalgae I obtained several unespecific bands but my plasmid control was perfect, can you give any advice on how to deal with this I'm thinking maybe increasing the Tm for the annealing.+

    Thx and great job with your research

    ReplyDelete