Team:Goettingen/NoteBook w10

PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration

-   Primers iGEM_67/68/69/70

1x reaction (50 µl)

         - preparation of master mix for 20 reactions containing

- addition of template (chrom. DNA/dH2O), primers and a part of the water (indicated in parenthesis in table for composition of each reaction) individually, then addition of 33 µl master mix

Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones

-   1.5 % Agarose-1xTAE gel

-   4 µl PCR reaction + 1 µl 5x DNA Loading Dye

-   5 µl of Test RDs; 1 µl uncut plasmid + 1 µl 5x DNA Loading Dye + 3 µl dH₂O

-   3 µl 2 log ladder

-   Run at ca. 100 V in 1xTAE buffer

-   Staining in EtBr and destaining in water

-    UV detection

Gel 1:

Gel 2:

-> RD: Expected band ca. 750 bp + ca. 2 kb for all digests. The expected bands were obtained for all RDs --> all clones contain plasmids --> prepare the plasmids for sequencing by G2L

-> PCR: for expected products: add ca. 2x 20 bp to sizes indicated above. The expected bands were obtained. However, for iGEM_67/68, iGEM_69/68 and iGEM_67/70, a slight band at approx. the bp of 2x expected was observed for 2 µl reaction (“product dimmers?”), this seemed to decrease with increasing primer amount. Hig iGEM_69/70 primer amounts seemed to interfere with PCR. The negative control only shows primer clouds at bottom of lanes. The optimal primer concentrations for the different PCR reactions seem to be:

Primers for generating reverse terminator insert

    -> Primers arrived: dissolved in HPLC water and diluted 1:20

-> Primers tend to form secondary structures --> Test PCR for optimal primer concentration

-> Annealing temperature: TA = 0.5 * [Tm(iGEM_71) + Tm(iGEM_72)] – 6 °C = 0.5 *[60 °C + 59.1] – 6 °C =53.55 °C

TEST PCR

1x reaction (50 µl) using part 7 C1 plasmid as template

Plasmid concentration when used as a template: 10 – 20 ng/ reaction

Part 7 C1 purified on 11.7.13; concentration = 120.5 ng/µl --> 1:10 dilution in water --> ca. 12 ng/µl

-  preparation of master mix for 5 reactions containing

-   addition of template (chrom. DNA/dH2O), primers and a part of the water (indicated in parenthesis in table for composition of each reaction) individually, then addition of 33 µl master mix

PCR protocol

-> run ON

Restriction digest of DarR (polyadenylated PCR product)

-> Volume: 12 µl of unpurified DarR polyA reaction

- Preparation of the digest:

-> 1 h at 37 °C in ThermoCycler

-  Purification of RD reaction using PCR clean up kit (Qiagen): 500 µl PB buffer, 1x elution with 22 µl pre-warmed HPLC water (incubation for ca. 4 min at RT, ca. 1 min at 50 °C)

-  Concentration (NanoDrop)

Purification of pBluescript(RD with EcoRI) from gel

-  1.1088 g (2 ml epi with gel) – 1.0376 g (another empty 2ml epi) = 0.0712 g --> ca. 70 mg gel --> 210 µl QG buffer were used

-  Elution with 2x 22 µl pre-warmed HPLC water

-  Concentration (NanoDrop)

Ligation of DarR/hybridization oligo inserts with pSB1C3/part6

-  Ligation reactions

A) DarR insert (from polyA product) + pSB1C3 E+P (from part 7; 31.7.13)

B) hybridization oligo Promoter 3-DarRoperator + pSB1C3 E+S (from part 7)

C) hybridization oligo Promoter 3-DarRoperator + part 6 E+ X

For DarR ligation: ratio 1:3 was kept --> ca. 50 ng vector (2070 bp) + ca. 43 – 51 ng insert (600-700 bp)

For Hyp oligo: 20- 30 ng vector + half amount of reaction mix from

-> Incubation at 16 °C for several hours (started at 11:30)

Gel run of DarR insert and pBluescript+EcoRI

-   1 % agarose-1xTAE gel

-    Loading of 3 µl 2 log ladder

-    Loading of 1 µl pBluescript (2.8.13; 342.0 ng/µl) + 3 µl dH₂O + 1 µl 5xLD

-    Loading of 4 µl pBluescript purified EcoRI RD + 1 µl 5xLD

-    Run at 100 V

-    EtBr staining + destaining in water

-    UV detection

Gel:

-> EcoRI-linearzied pBluescript seems to be pure. Still, shortly below the linearized-plasmid-band, some additional weak bands were seen --> possibly unspecific side products? But the strong band of the supercoil-plasmid is gone --> further digest with PstI

-> For DarR insert, an additional band at 0.1 kb was obtained --> polyA’s that were cut off by the enzymes??? Or a non-specific side product from PCR? --> if this fragment can be cloned, we will notice it when the transformants are tested in Colony PCR…

Digest of pBluescript-EcoRI with PstI

Ca. 33 µl pBluescript left

-> Addition of 4 µl FD buffer and 4 µl PstI FD; incubation for 1 h at 37 °C

Design of oligos for self-hybridization

-> These oligos contain the reverse complement sequence of the parts indicated below, since they are designed for reverse integration into pSB1C3 (DarR transcription unit)

-> Oligos hybridization leads to EcoRI and SpeI overhangs

-  For strong RBS (part 8)

-  For promoters parts 1,2,3,4 (an additional sequence of ca. 60 bp (reverse translation of “cyclicdiampacterim”) was added in front of the promoter so that binding of RNA-Pol does not interfere with binding of RNA-Pol to promoter of GFP transcription unit)

Transformation of parts from distribution kit 2013 (CFP, YFP)

Inoculation of 3 clones of each transformation in LBcm for miniprep

Preparation of back-up plates

-> Forgotten in fridge (4 °C) on 5.8.13

-> Put to 37 °C by Dominik today