PCR of the ribosomal gene


1. Subject

To execute a restriction analysis or sequencing, some parts of the ribosomal DNA is used. By means of so called primers a selected part of the ribosomal DNA is amplified. This reaction is called PCR (polymerase chain reaction).

2. Principle

The ribosomal DNA contains repeats, that consist of a small subunit (SSU, 18S),  an internal transcribed spacer (ITS1), 5.8S, an internal transcribed spacer (ITS2) and the large subunit (LSU, 26S). These repeats are separated by NTS, non-transcribed spacers.

3. Reagents and materials

3.1    electrophoresis-unit
3.2    bromophenolblue ([115-39-9] Biorad 161-0404)
3.3    primer NS1 (5'-GTAGTCATATGCTTGTCTC-3')
3.4    primer NS24 (5'-AAACCTTGTTACGACTTTTA-3')
3.5    primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3')
3.6    primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3')
3.7    primer ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3')
3.8    primer 5.8SR (5'-TCGATGAAGAACGCAGC-3')
3.9    primer LR7 (5'-TACTACCACCAAGATCT-3')
3.10  primer NS7 (5'-GAGGCAATAACAGGTCTGTGATGC-3')
3.11  primer V9G (5'-TTACGTCCCTGCCCTTTGTA-3')
3.12  primer LS266 (5'-GCATTCCCAAACAACTCGACTC-3')
3.13  ultrapure dNTP set 100 mM (BioLine, BIO-39025):
         2'-deoxyadenosine-5'-triphosphate, 2'-deoxythymidine-5'-triphosphate, 2' deoxycytidine-5'-triphosphate en 2'-deoxyguanosine-5'-triphosphate
3.14  DNA Taq-polymerase 5U/μl (BioLine, BIO-21040)
         The kit contains an ammonium buffer (10x) and MgCl2 (50 mM)
3.15  potassium chloride, KCl ([7447-40-7] Merck 4936)
3.16  Tris(hydroxymethyl)-aminomethane ([77-86-1] Fluka 93362)
3.17  MgCl2·6H2O ([7786-30-3] Merck 5833)
3.18  gelatine ([9000-70-8] Merck 4078)
3.19  Triton X-100 ([9002-93-1] Merck 8603)
3.20  mineral oil ([8042-47-5] Sigma M 3526)
3.21  1 N HCl [7647-01-0] (Sigma H-9892)
3.22  sodium hydroxide, NaOH ([1310-73-2] Merck 6498)
3.23  glycerol 87%, ([56-81-5] Merck 4093)
3.24  Na-EDTA [6381-92-6] (Titriplex III, Bio Rad 161-0729)
3.25  1,4-dithiotreitol, DTT ([3483-12-3] Sigma D-0632)
3.26  eppendorfcups 1.5 ml (Sarstedt 72.690)
3.27  Eurogentec SmartLadder (Eurogentec MW-1700-02)
             (200, 400, 600, 800, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, 10000 bp resp. 20, 40, 60, 80, 100, 15, 20, 25, 30, 40, 50, 60, 80, 100 ng/band)
3.28  PCR vials ultra thin 200 μl (Biozym 179401)
3.29  GelRed 10,000x in DMSO (Biotium 41002)

4. Solutions

4.1   PCR-buffer 10x:
Add 9.32 g potassium chloride [3.15], 3.03 g Tris [3.16] and 1.53 g magnesiumchloride hexahydrate [3.17] to 220 ml ultrapure water.
Adjust pH to 8.3 by adding ± 10 ml 1N HCl [3.21]. Add 0.25 g gelatin [3.18] and 2.5 ml Triton X-100 [3.19], heat the solution for 30 min. at 56°C.
Adjust the total volume to 250 ml with ultrapure water. Make aliquots of 50 ml.

4.2   Tris 1M, pH 8:
Dissolve 121.1 g Tris [3.22] op in 800 ml ultrapure water. Adjust pH to 8.0 by adding 1N HCl [3.27]. Add ultrapure water to a total volume of 1000 ml.
4.3   EDTA 0.5M, pH 8:
Add 186.1 g EDTA [3.24] to 800 ml ultrapure water and stir on a magnetic stirrer. Add while stirring ± 20 g sodium hydroxyde [3.22]. The pH will be approx. 8.0.
Adjust total volume to 1000 ml with ultrapure water.

4.4   DTT 1M:
Dissolve 1.54 g DTT [3.25] in 10 ml ultrapure water. Aliquot in portions of 1 ml and store at -20°C.
4.5   dNTP-mix 5mM:
Mix 50 μl dATP, 50 μl dTTP, 50 μl dCTP, 50 μl dGTP [3.13] and 800 μl ultrapure sterile water into a sterile eppendorfcup (3.26). Make aliquots of 50 µl and store at -20°C.
Before use, dilute this solution to 1 mM by adding
200 μl ultrapure sterile water to an aliquot.
4.6   Taq polymerase dilution buffer:
Mix 0.2 ml 1M Tris, pH 8.0 [4.2], 0.2 ml 0.5M EDTA pH 8.0 [4.3], 0.1 ml 1M DTT [4.3], 49.5 ml ultrapure water and 50 ml glycerol 87% [3.23] thoroughly. Store solution at 4°C.
4.7   Taq polymerase user solution 1U/µl:
Mix 20 μl DNA-polymerase 5 units/μl [3.14] with 80 μl DNA-polymerase dilutionbuffer [4.6] and vortex. Store solution at -20°C.
4.8   Loading buffer:
Dissolve 50 mg bromophenolblue [3.2] in a few drops of ethanol. Mix 5 ml glycerol 87% [3.23] with 45 ml ultrapure water. Add the dissolved bromophenolblue.
Mix both solutions and store at 4°C
. Before use prepare vials with 1 ml loadingbuffer and add 3 µl GelRed [3.29].
4.9   TE-buffer, pH8:
Add 0.12 g Tris [3.16] and 0.04 g Na-EDTA [3.24] to 80 ml ultrapure water. Adjust pH at 8.0 with 1N HCl [3.21]. Heat the solution if EDTA doesn’t dissolve very well.
Adjust volume to 100 ml. Autoclave the solution for 15 min. at 121°C. Store at roomtemperature.

4.10  SmartLadder:
The SmartLadder of Eurogentec (3.27) is user ready. Per lane 5 μl is used. With this marker the size and the concentration of the amplicon can be estimated.
It is possible to use 2.5 µl and still have visible marker bands. The concentrations of the bands have to be divided by 2.
4.11  Primer stock solution:
Primers are usually sent in a lyophilized state. Resuspend the primer in sterile, ultrapure water to obtain a concentration of 100 pmol/µl.
Before use, 10 µl stock solution is diluted 10x by adding 90 µl sterile, ultrapure water. The final concentration is 10 pmol/µl.

5. Protocol

The PCR can be performed with the distributed buffer [3.14] or with the 'home-made' buffer [4.1]. See tables [8.1] and [8.2] respectively.

 

5.1  Total PCR mastermix for N samples + blank [6.1].
5.2  Pipet 1 µl DNA extract in a PCR vial [3.28]. Add 24 µl of the mastermix [5.1].
5.3  Run a PCR program according to primer combinations [8.3].
5.4  Check the results of the PCR by performing an electrophoresis on agarose-gel by mixing 2.5 µl PCR product and 5 µl loading buffer [4.8].
5.5  A reference ladder, e.g. SmartLadder [4.10] is also mixed with 5 µl loading buffer [6.2].

6. Remarks
6.1   It is wise to add 10% extra samples. E.g. when making a mix for 50 samples use a multiplication facter of 55.
6.2   In this case you might also use 2.5 µl SmartLadder; the bands will still be visible and the volume for electrophoresis is the same as the samples.

7. Literature
7.1   Protocol Online
7.2   Sambrook et al.

8. Tables and graphs
8.1   PCR mastermix using ammonium buffer (10x):

PCR mix volume for 1 sample
Water 14 µl
NH4+-buffer (10x) 2.5 µl
dNTP (1 mM) 2.5 µl
Mg2+ (50 mM) 1.0 µl
Primer 1 (10 pmol/µl) 1.0 µl
Primer 2 (10 pmol/µl) 1.0 µl
DMSO 1.0 µl
Taq polymerase (0.5U) 1.0 µl
Total volume:  24 µl


8.2   PCR mastermix using buffer [4.1]:

PCR mix volume for 1 sample
Water 16 µl
PCR buffer (10x) [4.1] 2.5 µl
dNTP (1 mM) 2.5 µl
Primer 1 (10 pmol/µl) 1.0 µl
Primer 2 (10 pmol/µl) 1.0 µl
Taq polymerase (0.5U) 1.0 µl
Total volume:  24 µl


8.3   PCR program based on the used primer combinations:

PCR cycle Temperature in °C Time in min:sec
Initial denaturation 94-98 2:00-10:00
First cycle step: denaturation 94-98 0:30-1:00
Second cycle step: annealing

V9G-LS266
ITS1-ITS4
NS1-NS24
Oli4-Oli16

50-56
48-55
48-52
48-52
0:30-2:00
Third cycle step: elongation 68-72 1:00-5:00
Final elongation 68-72 2:00-10:00


A normal PCR is performed with 25-30 cycles. E.g. a PCR of ITS1-ITS4 looks like this:

Initial denaturation: 95°C for 5 min.

35 cycles:
Denaturation 95°C for 45 sec.
Annealing 55°C for 30 sec.
Elongation 72°C for 1 min.

Final elongation: 72°C for 6 min.
Cooling to 15°C