Team:Freiburg/Notebook/7 September

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Commons

Ligation

Investigators: Sandra

Ligation of PR1-PR6 with RFP (S1b and S2a). PR1-PR6 were digested with antarctica before start of ligation. Start of ligation at 12:40. Incubation at room temperature for at least 3 hours.

Trafo

Investigators: Sandra

Trafo of:

• PR1+GFP
• PR2+GFP
• PR3+GFP
• PR4+GFP
• PR5+GFP
• PR6+GFP

• PR1+50b
• PR2+50b

PCR of psB1C3

Investigators: Julia

blue light receptor

no colonies on the plates

There were no colonies on the plates with ligated parts of the 2A-assembly.

Miniprep

Investigators: Sophie
yesterday two tubes with LB were inoculated with cells from our S35 glycerol stock(BBa_K322999). Today minipreps were performed.

PCR

Investigators: Sophie

PCR-Mixture for one Reaction:

For a 50 µl reaction use

Digestion with Dpn I and purification with PCR purification kit What program do you use?

First 20 cycles touchdown 69°C -0.4/cycle

next 10 cycles touchdown 72°C -0.2 per cycle

PCR products were loaded onto a gel

Lysis cassette

Troubleshooting of the modified Lysis genes K124017

Investigators:Theo

S4+S15 Nr1 and 7 were digested with XbaI and PstI, loaded on a 1% agarose gel and extracted on the 6th of September.

Today I digested the quickchanged-temperature sensitive promotor (K098995) with SpeI and PstI for ca 3 hours, incubated it with antarctic phosphatase for 1 hour and PCR-purified it.

PCR Purification and Gel documentation

PCR Purification Kit

Qiagen Kit

Procedure

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary

to remove mineral oil or kerosene.

For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).

1. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow.

If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH

5.0, and mix. The color of the mixture will turn to yellow.

1. Place a QIAquick spin column in a provided 2 ml collection tube.
2. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
3. Discard flow-through. Place the QIAquick column back into the same tube.

Collection tubes are re-used to reduce plastic waste.

1. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
2. Discard flow-through and place the QIAquick column back in the same tube.

Centrifuge the column for an additional 1 min.

IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless

the flow-through is discarded before this additional centrifugation.

1. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
2. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to

the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively,

for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick

membrane, let the column stand for 1 min, and then centrifuge.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved

between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

1. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to

5 volumes of purified DNA. Mix the solution by pipetting up and down before

loading the gel. Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and orange G) that facilitate estimation of DNA migration distance and optimization of agarose gel run time. Refer to Table 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.

Documentation:

Why are you doing this experiment? Name the parts which you cut out.

Insert Gel Picture here and mark the bands you will excise. Describe the used ladder and sizes of the bands.

Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results. Also make a note of the ratio between A260/A280 for each sample.

Ligation and Transformation

The amount used for the gel was 5mikrol, so this corresponds to about 60ng DNA from the M48 sample.
One can see that the concentration of M66 cut with EcoRI and PstI is about 2-3 times higher (cannot be measured because of the agarose from the gel extraction).
The concentration of S4+S15 Nr1 should be about 1/2 of that of M48, so I am going to use 2x and 6x the amount of M48 for the ligation.
There is no band to be seen for S4+S15 Nr7, so the concentration is lower than 5ng/mikrol, so I am going to use all of it for the ligation (ca 25mikrol) and change the reaction concentrations accordingly.

Procedure

PCR tube:

total volume 20 μl

1. add H2O                                 (17 μl -X-Y-Z)

2. add 2 μl Ligase Buffer 10x

3. add Insert   

4. add Vector                           (20ng needed)

5. Add 1 μl T4-DNA Ligase

6. Incubate  10-30 min at room temperature

Precipitator

Trafo

Investigators: Sandra

Trafo of the already ligated parts of the pbd + Gst-vector

Ligation

Investigators: Sophie

For pbd in iGEM C3-vector: again Ligation (see 31.08.11) because I don't find the last ligation product that produced positive clones. I have to redo the Trafo because the last positive clones seem to have mutated over the last generations, probably because the pbd is toxic.
labelled: L S54-ε5-C3 1:1 / 1:3
stored in "Ligationsreaktionen"-box