Team:Freiburg/Notebook/lab crrna plasmid

Effector

Effector Control

Targeting

Multiple targeting

crRNA-Plasmid

August

September

GFP-reporter-plasmid

uniBAss

Modeling

Standardisation

Protocols

Labbook RNA plasmid - pIG0021

As a central part of our toolkit we need to clone a plasmid that contains a tracrRNA and a locus for inserting CRISPR RNAs into the PSB1C3 backbone. The general strategy is to amplify both loci from the px334a plasmid by Feng Zhang and combine them in the iGEM backbone. iGEM biobrick cutting sites will allow us to combine several targets with no big effort.

August

28.08.13

All primers arrived, PCRs for fragments.

pU6:crRNA is amplified from px334a by PCR using the primers oIG0056 and the oU6:crRNA_fw primer. pH1:tracrRNA is amplified from pX334a by PCR suing the primers oH1:tracrRNA_fw and oH1:tracrRNA_rev.
PCR was performed using the following master mix and reaction conditions. In the same reaction the backbone of PSB1C3 was amplified using the primers oIG0037 and oIG0038.

• Annealing: 60°C
• Elongation: 120 sec
• 24 cycles

Expected sizes are ~500bp for the RNA fragments and 2kb for the backbone.
Somebody must have mixed up TAE and ddH2o by preparating agarose. The gel was not really resolving the DNA. Nevertheless, the fragments contained the approximate size fragments. Bands were cut out and extracted using the Roche GelEx kit.

Yields:
pH1:tracrRNA: 2.2ng/ul
pU6:crRNA: 5.6 ng/ul
backbone: 22.1 ng/ul

Fusion PCR with all three fragments

As earlier approaches showed these fragments are problematic when using multi-fragment Gibson cloning or classical cloning respectively. Therefore the fragments were ligated by fusion PCR.

first PCR

• Annealing: 60°C
• Elongation: 180 sec
• 6 cycles

second PCR

1ul of oH1:tracrRNA_fw and oIG0038 were added for complete amplification of linearised RNA plasmid.

• Annealing: 60°C
• Elongation: 180 sec
• 24 cycles

This was done as a duplicate and put on a 1% agarose gel. The expected band runs at 3kb.

Roth 1kb ladder was loaded. There are three bands visible, but only the most upper band fits the expected size. This band was cut out in both lanes and extracted by the Roche GelExtraction kit.
Yields
lane1: 9.6 ng/ul
lane2: 19.6 ng/ul

Gibson assembly for pIG0021

As the extracted linearised RNA plasmid contains complementary overhangs a 1-fragment Gibson was performed. To an aliquot of Gibson master mix 2.5 ul, respectively 5 ul of DNA was added and filled up with ddH2O to 5 ul. This mixture was incubated on 50° for 1 hour. Afterwards it was incubated for 3 minutes on ice and 3 minutes on RT.
5ul of this mixture was transformed into chemically competent E.coli and spread on chlorampheicol containing Agar plates.

29.08.2013

Colony PCR

Clones were obtained. Colony PCR was performed to screen for potential positive clones. 15 clones were picked, transferred to Master Mix, transferred to Master Plate.

cPCR pIG0021

Results of test digest

6 out of 8 colonies were positive. Clone 2 and 4 were sent for sequencing with oIG6017.