24 lane gel showing presence or absence of bacterial DNA.

 

Lanes with pink bands indicate presnence of DNA in our ssample. These samples were then selected to proceed through the next steps of DNA purification.

Here's the science...

PCR 1

1. Place the following substances in a 0.2ul Eppendorf tube:
Various amounts of pond, stream and mouth samples (See table below)
One PCR bead containing:Taq, DNPT, DdNPT, Buffer(10ul)
16s Forward Primer
16s Reverse Primer
Template (DNA)
BSA (Bovine Serum Albumin) in some tubes (see table below).

2. Place tubes in PCR machine, having a good system to keep track of each tube.
Use #25 protocol for the PCR machine:
95C for 5 min.
94C for 45s )
50C for 45s } 35X
72C for 90s )
72C for 7 min.
4C until ready for use

PCR Reaction Elements
16s Forward Primer (10ul) 1 1 1 1 1 1 1 1 1
16s Reverse Primer (10ul) 1 1 1 1 1 1 1 1 1
Template (DNA) 1 5 10 1 5 10 1 5 10
BSA (10mg/ml) 0 0 0 5 5 5 10 10 10
Water 22 18 13 17 13 8 12 8 3

Total Volume (25 ul) 25 25 25 25 25 25 25 25 25

16s Forward Primer: Bac16sF (5’-CCGAATTCGTCGACAAGAGAGTTTGATC-3’)
16s Reverse Primer: Bac 16sR (5’-CCCGGGATCCAAGCTTACOGCTACCTTG-3’)

Gel Electrophoresis (Screening)

1. Prepare a 1% agarose solution (using TBE buffer).
2. Place a 2ul drop of loading dye on parafilm for each well.
3. Add 5ul of PCR product to each dot and load the gels.
4. Run them for about 30 minutes.
5. Place them in a staining container, cover with ethidium bromide and stain for 10 minutes.
6. Remove the ethidium bromide and cover with distilled water for 5 minutes.
Place the gel under uV light and review the results.

Bacteria Cloning

1. Combine:

PCR product (0.5ul and 2ul) (See table below)
1mMl salt solution (CaCl )
+ n H O
5ul total volume
+ 1ul vector (TOPO)
6 ul cloning reaction
2. Incubate on ice for 30 minutes.

Heat shock tubes for 30 seconds at 42 C. (Do not shake.)

3. Mix and incubate at room temperature for 5 minutes.

4. Thaw E. coli tubes.

5. Add 1ul TOPO cloning reaction to E. coli cell vial.

6. Incubate on ice for 30 minutes.

7. Heat shock for 30 sec. at 42C. Do not shake.

8. Add 250 ul SOC, cap and shake 1 hour at 37 C. (Shake gently every 15 minutes.)

9. Spread 50ul SOC mixture on one set of bacterial plates (made with imMedia Amp Blue) and spread 200ul of the SOC mixture on another set of plates.

10. Incubate overnight and choose the white colonies.

Amt. Of PCR Product
Pond (A2) 0.2 um filter .5 ul PCR

2 ul PCR

(B5) 1.2 um filter .5 ul PCR

2 ul PCR

Stream (C6) 0.2 um filter .5 ul PCR

2 ul PCR

(D5) 0.2 um filter .5 ul PCR

2 ul PCR

Mouth (M4) 0.2 filter .5 ul PCR

2 ul PCR