Amplyfing of Bax sequence

Justyna

Methods

• PCR reaction mix:

per 50μl:

5.0 μl - 10 x buffer (20mM MgSO4)
3.5 μl - dNTP mix (5mM)
3.0 μl - primer BaxF (0.5 μM)
3.0 μl - primer BaxR2 (0.5 μM)
1.0 μl - template DNA
32.0μl - mQ water
2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)



• Template DNA in 3 dilutions - 10¹ (pure plasmid DNA), 10^-1 and 10^-2.
• buffer, dNTP mix and polymerase from EURx.
• DNA template - human Bax with changed codons to be more efficient in yeast.





Thermal cycling conditions for PCR (~7-8 kb):




94°C, 1 min 0s

94°C, 0 min 15s
69°C-74°C, 0 min 30s
68°C, 1 min 0s
cycles 1-10

94°C, 0 min 15s
69°C-74°C, 0 min 30s
68°C, 1 min 20s
cycles 11-25

68°C, 7 min 0s
4°C, indefinite




Results:

• Best reaction for 10¹ and 10^-1 (more specific), in 69.1°C and 69.8°C.
  
  
  
  
  
  
  

Cloning of the mgtc promoter into the pSB1A3 plasmid

Kamil

Tasks:

• Reamplification of the mgtc promoter

Methods:

• The PCR mix was prepared as follows:

  5μl buffer B (EURx)
   2μl 5mM dNTPs (EURx)
   5μl forward starter
   5μl reverse starter
   2μl OptiTaq polymerase (EURx)
   2μl matrix 
   29 μl H2O 

• PCR programme:

   4min 95°C 
   (30s 95°C, 35s 58°C, 40s 72°C)x28 
   10min 72°C 
   ~ 4°C
  

• The results were visualised with gel electrophoresis on 1% agarose gel.
• The appropriate bands were extracted using Gel-Out kit (A&A Biotechnology) according to the producers protocol.

Results:

• Gel Electrophoresis:

From left:

1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. 50μl of PCR mix
3. 50μl of PCR mix
4. Negative control

Conclusions:

• Reamplification was successful.

Assembly of endosomal detection operon

Marcin

• Task 1: Gel-out of samples which was cut out from the gel in 12.08.09

Procedure:

• Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here

Comment:

The concentration of DNA is sample containing BBa_R0080 was surprisingly low. Because of this I decided to repeat the digest and try to use another gel-out kit.

• Task 2: Restriction digest of BBa_B0032

Methods:

• Digest of BBa_R0080 using SpeI and PstI
• Reaction mixture composition:

 
20 μl purified plasmid DNA product
0.5 μl SpeI (Fermentas)
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Task 3: Cloning the sequences: cro CDS and BBa_E0022 with BBa_B0032 vector

Methods:

• Ligation mixtures composition:

 
15 μl digested cro (or E0022) 
8 μl digested vector 
3 μl Tango buffer(Fermentas)
3 μl dNTPs mixture (EurX, concentration 5 mM) 
1 μl ligase T4 (Fermentas)

• Duration of ligation was about 18 hours; reaction was conducted in 19 °c (approximately).
• In the next step ligated samples were thermally inactivated via heating in 80 °c for 20 minutes

Task 4: Prepare the bacterial cultures for isolation of on pSB1A3 plasmid containing:

• p53
• BBa_B0032+BBa_C0040
• BBa_B0032+BBa_C0051
• BBa_B0032+BBa_E0032