PCR PhoP and PhoQ

Kama

Tasks:

• Amplification of phoP and phoQ

Methods:

• PCR mixture's composition:

  1μl Pfu buffer (EURX) 
  0,25μl primer phoPF for phoP and phoQF for pho Q
  0,25μl primer phoPR for phoP and phoQR for pho Q
  0,5μl dNTPs (10 mM)
  0,25μl Pfu turbo polymerase (EURX)
  0,5μl template DNA from Salmonella (Salmonella enterica s. Thypimurium LT2)
  0,75μl DMSO 
  The solution was topped up with H2O to 10μl.

• PCR programs:

phoP

4min 95°C 
 (30s 95°C, 1min 60°C, 1min 40s 72°C)x35
 10min 72°C 
 ~ 7°C

phoQ

4min 95°C 
 (30s 95°C, 1min 60°C, 3min 10s 72°C)x35
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Assembly of fusion proteins

Marcin

Task 1: Amplification of p53 (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition:

5 μl Pfu buffer (EURX) 
2 μl Pfu turbo polymerase (EURX)
5 μl dNTPs (EURx, 5 mM)
0,1 μl template DNA (isolated plasmid)
1 μl primer p53silverF
1 μl primer p53silverR
36 μl MQ water

PCR program:

1. 98°C - hold
2. 98°C - 2 minutes
3. 98°C - 15 sec
4. 64°C - 30 sec
5. 68°C - 2 minutes
   go to 2 until number of cycles=35
6. 68°C - 10 minutes
7. 4°C - hold

Task 2: Amplification of bax (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition:

5 μl Pfu buffer (EURX) 
2 μl Pfu turbo polymerase (EURX)
5 μl dNTPs (EURx, 5 mM)
0,1 μl template DNA (isolated plasmid)
1 μl primer bax_silverF
1 μl primer bax_silverR
36 μl MQ water

PCR program:

1. 98°C - hold
2. 98°C - 2 minutes
3. 98°C - 15 sec
4. 70°C - 30 sec
5. 68°C - 1.5 minutes
   go to 2 until number of cycles=30
6. 68°C - 5 minutes
7. 4°C - hold

Task 3: Amplification of signal peptide from COX (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition:

5 μl Pfu buffer (EURX) 
2 μl Pfu turbo polymerase (EURX)
5 μl dNTPs (EURx, 5 mM)
0,1 μl template DNA (isolated plasmid)
1 μl primer mitoCOXsilverF
1 μl primer mitoCOXsilverR
36 μl MQ water

PCR program:

1. 98°C - hold
2. 98°C - 2 minutes
3. 98°C - 15 sec
4. 69.3°C - 30 sec
5. 68°C - 45 sec
   go to 2 until number of cycles=30
6. 68°C - 5 minutes
7. 4°C - hold

Task 4: Amplification of YFP (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition:

5 μl Pfu buffer (EURX) 
2 μl Pfu turbo polymerase (EURX)
5 μl dNTPs (EURx, 5 mM)
0,1 μl template DNA (isolated plasmid)
1 μl primer YFPsilverF
1 μl primer YFPsilverR
36 μl MQ water

PCR program:

1. 98°C - hold
2. 98°C - 2 minutes
3. 98°C - 15 sec
4. 70°C - 30 sec
5. 68°C - 1.5 minutes
   go to 2 until number of cycles=30
6. 68°C - 5 minutes
7. 4°C - hold

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Plasmid isolation
• Control digest

Methods:

• The plasmids were isolated from 3μl of overnight liquid cultures using the Plasmid Mini kit (A&A Biotechnology).
• The digest mix was prepared as follows:

  20μl purified plasmid 
  3μl Orange Buffer (Fermentas)
  1μl PstI Enzyme (Fermentas)
  1μl EcoRI Enzyme (Fermentas)
  5μl H2O

• The digest was carried out in 37°C for 4h.
• The results were visualized on 2% agarose gel.

Results:

• Gel (from left):
1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. Sample A
3. Sample B
4. Sample C
5. Sample D
6. Sample E
7. Reference sample
8. Sample F
9. Sample G
10. Sample H
11. Sample I

Conclusions:

• Sample F looks like a hit. It was sent to be sequenced.