Team:Evry/Notebook/Interlabstudy/week8

From 2014.igem.org

Week 8

Interlab Study

08.18.2014

Sequencing results were received for BBa_J23101 (26DJ62 and 26DJ63) and BBa_J23115 (26DJ64 and 26DJ65) PCR products.
26DJ62

TCAGANNAAAAAAATCCTTAGCTNNCGCTAAGGATGANNTCTGGAATTCGCGGCCGCTTCTAGAG'''TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC'''TACTAGTAG CGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTC GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAGAG

26DJ63

GAGCGCANCGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTG CAGCGGCCGCTACTAGTA'''GCTAGCATAATACCTAGGACTGAGCTAGCTGTAAA'''CTCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTT TTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGCAAGGTGGCAAA

26DJ64

TCAGANNAAAAAAATCCTTAGCTNNCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG'''GTTTATAGCTAGCTCA[[T]]CCT[[A]]GGTACAATGCTAGC'''TACTAG TAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGAC TCGCTGCGC TCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAGG

26DJ65

CNGCGAGTCAGTNAGCGAGGAAGCCTGCANNACGCGAAGTNATCTTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTNGCCCTTTTTTGCCGGACTGCAGC GGCCGCTACTAGTA'''GCTAGCATTGTACCTAGGACTGAGCTAGCTATAAA'''CTCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTA TCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAAC

The mapping was perfect for BBa_J23101. There were 2 mismatches for BBa-J23115, one at 17 and the other t 21 on the promoter sequence that correspond to the K823012 as mentioned on 2014.igem.org/Tracks/Measurement/Measurement_Worksheet. The mapping was perfect between sequencing reads and K823012 sequence was perfect.

Colony PCRs were performed on 8 colonies for BBa_E0240 and BBa_I20260, form plates from the transformation of 16th August.

Table 3: PCR mix Preparation for One Taq PCR colony

49 µl of mix were distributed in each PCR tubes. The One Taq PCR program was applied.

Table 4:IGEM One Taq PCR program termocycling conditions

08.19.2014

Preparation of a 1% agarose gel: 1.01 g of Top Vision agarose (Thermo Scientific) + 100 ml of TAE 1X. Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders. Gel running 45 minutes at 100 mV in TAE 1X buffer.

Figure 6: 1% agarose gel of colony PCR products for BBa_E0240 and BBa_I20260. Lane 1 and 10: Purple 2-Log ladder NEB

We decided to amplify the colony 1 for BBa_E0240 and colony 2 BBa_I20260. So PCR products were purified via with the GeneJET purification kit (Thermo Scientific)followed by DNA quantification with the NanoDrop 2000 (Thermo Scientific). BBa_E0240 purified PCR product: 61 ng/µl and BBa_I20260 purified PCR product: 37.8 ng/µl.
Purified PCR product aliquot was send to sequencing. The sequencing number were 26DJ68, 69, 70, 71.
Preparation of miniprep culture of BBa_I20260 and BBa_E0240 selected colonies in 5 ml of LB with respectively 5 µl of Kana or chloram solution stock.Incubattion overnight at 37°C and 300 rpm.

08.20.2014

Miniprep cultures from the 19th August was purified with the NucleoSpin Plasmid kit (Macherey-Nagel). DNA was quantify with the Nanodrop 2000. DNA concentrations and A260/280 were respectively for BBa_E0240 and BBa_I20260: 42 ng/µl and 1.91 ; 13.7 ng/µl and 1.71.

08.21.2014

Transformations of registry parts (BBa_J23115 and J23101) were performed to obtain colonies with plasmid containing parts for future amplifications.
The transformation was performed on DH5 alpha ''E. coli'', as followed:

Remove E. coli competent tubes from -80°C and keep it on ice
Add 3 µl of template (here solubilized plasmids from the registry distribution kit) and mix gently
Incubate 10 minutes on ice
Perform an heat shock 30 seconds at 42°C
Incubate 2 minutes on ice
Add 2 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
Plate 200 µl of BBa_J23115 (K823012) on a chloramphenicol LB agar plate or ampiciline for BBa_J23101
Incubate plate overnight at 37°C

08.22.2014

Transformation plate observation:
- BBa_J23101: nothing grown
- BBa_J23115 (K823012): 100-150 isolated colonies

A PCR was performed on 8 colonies for BBa_J23115 (K823012) following the protocol Table 3 and 4.