Gradient PCR Pho

Kama

Tasks:

• Amplification of phoP/phoQ

Methods:

• PCR mixture's composition:

  1μl pfu buffer (Fermentas) 
  1μl MgSO4 (Fermentas) 
  0,5μl primers 
  0,5μl dNTPs (10 mM)
  0,25μl pfu turbo polymerase
  0,5μl template DNA from Listeria??? (Salmonella)
  optionally: 0,75μl DMSO

  The solution was topped up with H2O to 10μl.

• PCR programs:

pho

4min 95°C 
 (30s 95°C, 1min 45-55°C, 4min 72°C)x3 
 (30s 95°C, 1min 55-60°C, 4min 72°C)x28 
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Quality check (cro PCR product and pKS isolate)

Kuba

• pKS-CRO cut with XbaI and EcoRI

• Gel (from the left)

1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. uncut plasmid (isolate no.1)
3. cut plasmid (isolate no.1)
4. uncut plasmid (isolate no.2)
5. cut plasmid (isolate no.2)
6. uncut plasmid (isolate no.3)
7. cut plasmid (isolate no.3)

Note: isolate no.1 contains a correctly inserted PCR product

Cloning of p53 coding sequence

Marcin

Comment:

There were some difficulties with ligation p53 coding sequence into pKS plasmid so I decided to take another sample containing isolate amplified p53 (via PCR reaction) and perform digest of the sample using XbaI

Tasks:

• Restriction digest of p53 coding sequence obtained from PCR reaction

Methods:

• Reaction mixture composition

22.5 μl PCR product (DNA concentration about 6.5 ng/μll) 
2.5 μl Tango Buffer (Fermentas) 
0.5 μl XbaI (Fermentas)

• Digest program:

Digest

 3h 37 °C

15 min 80 °C

~4 °C

• Quantification of the amount of DNA after digest on the agarose gel

Methods:

• Electrophoresis of the digested DNA sample:

Conditions:

agarose concentration - 1%

voltage - 70V

time - about 30 minutes

After electrophoresis gel was irradiated with UV light and photographed:

Comment:

The sample must have degraded after gel-out procedure. Most probably due to acidic condition in the solution DNA hydrolysed. It is obligatory to do another PCR reaction using the old plasmid sample (I hope the DNA is not degraded!).

Miecznikowa team Aim: debug RFP-terminator ligation that did not work

Jarek/Franek/Ania

Task1:

• Alkaline lysis of the plasmid containing Terminator

Methods:

• PlasmidMini set by A&A Biotechnology was used. 2 test tubes with 5ml LB and 5 ul Ampicyline were first inoculated with the plasmid containing colonies. The cultures were incubated over night at 37C.Alkaline lysis was performed on both cultures. The pellet from 5 ml of bacteria was used.

Results:

• DNA extraction quality controll.DNA Concentration was measured using Spectrophotometer NanoDrop ND-1000

Task2:

• We decided to measure the concentration of DNA in our samples for future use e.g. efficient ligation mix. NanoDrop ND-1000 was used.

Results:

Notes - IMPORTANT:

• Rfptra and RBSdigested samples were extracted from the gel using gel-out kit by A&ABiotechnology. Presented data shows that after gel extraction there is very little DNA in the sample.
  
  Probably there is a ****problem with gel extraction procedure**** - the Gel-out set by A&A Biotechnology might be defective.
  
  

Today the intermediate bricks were designed and we divided the tasks as follows:

• Franek: BBa_J5528, digestion with (EcoRV, PstI) and insertion into pKS2 (Bluescript) cut with SmaI i PstI (look for white colonies on the X-gal, IPTG, Amp plates)
• Jarek: construct BBa_K177011
• Michał: construct BBa_K177012
• Marek: construct BBa_K177013
• Ania: construct BBa_K177014
• Monika: transform competent cells with BBa_K1763004, BBa_S03473, BBa_E0840, BBa_J07037 out of the distribution.

potential problems: get inv/llo/phoP/phoQ