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week number 25

▼2015-06-19 Quantitative Gel of the PCR of the Gibson assembly fragments

Description:

We performed a gel quantification of DNA yielded in the PCR of our Gibson fragments.

 

Procedures:

 

Quantitative DNA gel:

 

Description:

 

Steps:

  1. Prepare an evenly stained agarose gel.
  2. Load a defined amount of the sample
  3. Load the DNA Ladder according to manufacturers protocol (e.g. NEB 2-log: 10µl for ng values according to sheet)
  4. Document and analyze the gel

 

Notes:

 

DNA samples were prepared by 1 hour of digestion with Dpn1 at 60 °C, followed by purification using a qiagen kit.

Samples were mixed with 3 parts of 6x loading dye, and loaded together with 2 log ladder.

Samples were: 1/2: PCR product of mCherry

3/4: CFTR testing construct

5/6: GFP

7/8: p413 backbone

Samples were yielded from PCR with Phusion polymerase.

 

Results summary:

Lane

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

DNA

2-log

mCherry

CFTR

GFP

P413

2-log

Basepairs

 

~800

200-300

~800

~6000

 

DNA Yield

 

~60 ng/µl

~60 ng/µl

~60 ng/µl

~60 ng/µl

 

 

Conclusion:

The bright bands yielded in this gel, in comparison to the previous quantitative gel of the PCR with OneTaq are indicative of a higher suitability of Phusion polymerase for constructs of this composition. Especially backbone p413 yielded a bright band under UV light in our present experiment, whereas in the previous experiment, there were no bands at all. This indicates, that we should choose Phusion for the amplification of larger fragments.

In the following quantification, all the bands yielded a concentration of about 60 ng/µl, fairly sufficient for a following Gibson Assembly.

▼2015-06-21 Colony PCR of E.coli transformated with Gibson-Plasmids

Procedures:

 

PCR:

 

Description:

 

        Materials and Chemicals:

 

        PCR-tubes

        Forward primer

        Reverse primer

        Mastermix (dNTPs, Polymerase, buffer)

        Water

        Thermocycler

 

        Endvolume: 10 µl

 

Steps:

  1. Pick colonies from plates. Solute one colony in about 20 µl of water.
  2. Give concentrated mastermix (which should be diluted to 1x in the end (e.g. you need 5 µl of 2x mastermix for 10 µl)), primer (concentration between and µl). Fill the volume to 10 µl with the water with E.coli
  3. Use the Thermocycler with the usual PCR program for at least 25 cycles.
  4. If the result is positive, take the remaining water to seed LB-medium with our E.coli

 

Notes:

 

        Plasmids: p413-GPD

        Insert: mCherry-CFTR-Tags-GFP

 

        Chemicals(one tube):

        0,4 µl forward Primer

        0,4 µl reverse Primer

        5 µl Mastermix

        4,2 µl water with E.coli

 

Results:

 

Culture 10 seems to be the only picked culture wich exhibits an insert

 

Figure 1: Colony PCR of E.coli transformated with Gibson-Plasmids

Positive Colonies exhibit an insert with the CFTR-testconstruct that is about 1,5 kb long.