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Team:Goettingen/NoteBook w-2
From 2013.igem.org
Redo the PCR of yesterday
Redo the PCR of yesterday
Re-running of PCR. This time with 4 L of 5 M forward and 4 L of 5 M reverse primers. Rest of the parameters are the same.
|
Components |
Volume (uL) |
|
Forward primer (5 M) |
4 |
|
Reverse primer (5 M) |
4 |
|
dNTPs (12.5 M) |
2 |
|
L. monocytogenes complete DNA |
2 |
|
5X HF Buffer |
10 |
|
PfuS (polymerase) |
1 |
|
HPLC H2O |
27 |
|
TOTAL |
50 |
Amplification seen for iGEM2 and 3 primers (approx. 600 bp). It is the cyclase domain (CD).
|
1 2 3 4 5 6 7 8
|
1: 100kb ladder 2: Primer iGEM 1 and 2 used 3: Primer iGEM 1 and 2 used 4: Primer iGEM 3 and 2 used 5: Primer iGEM 3 and 2 used 6: positive control 7: one Primer used 8: 1kb ladder
|
PCR to clone dacA from genome
PCR to clone dacA from genome
|
component |
Sequence (5’-3’) |
|
T7 Promoter |
TAATACGACTCACTATAGGG |
|
T7 Terminator |
GCTAGTTATTGCTCAGCGG |
We have T7 Promoter on one side and T7 Terminator on the other side of the multiple cloning sites. iGEM 1 has restriction site for SacII at 5’ end. iGEM 2 has restrictions site for SacI at 3’ end. iGEM 3 has restriction site for SacII at 5’ end. iGEM 1 and 2 yields a product of roughly 900 bp (full length dacA). iGEM 2 and 3 on the other hand yields roughly 600 bp (cyclase domain).
PCR constituents
|
Components |
Volume (L) |
|
Forward primer (5 M) |
2 |
|
Reverse primer (5 M) |
2 |
|
dNTPs (12.5 M) |
2 |
|
L. monocytogenes complete DNA |
2 |
|
5X HF Buffer |
10 |
|
PfuS (polymerase) |
1 |
|
HPLC H2O |
31 |
|
TOTAL |
50 |
PCR extension time = 1 min
M 1 2 3 4
Lane 1,2 : Listeria with primer iGEM 1 and 2
Lane 3,4 : Listeria with primer iGEM 3 and 2
Plasmid prep of pGP172 and digestion with SacI and SacII separately
Plasmid Isolation (pGP172)
We isolated pGP172 plasmid from E. coli (4 mL overnight culture) in two batches
Qiagen® kit method (mini prep)
|
Batch Number |
Concentration (ng mL-1) |
|
1 |
49.6 |
|
2 |
49.4 |
Restriction Digestion of pGP172 with SacI and SacII separately
|
Components |
Volume (L) |
|
1500 ng plasmid |
20 |
|
Fast Digest® SacI |
4 |
|
Fast Digest® buffer |
4 |
|
HPLC H2O |
12 |
|
TOTAL |
40 |
Fast Digest® SacI incubation at 37 oC for 2 hours
|
Components |
Volume (L) |
|
1500 ng plasmid |
20 |
|
SacII |
4 |
|
Buffer |
4 |
|
HPLC H2O |
12 |
|
TOTAL |
40 |
Fast Digest® SacII incubation at 37 oC for 5 hours
After digestion, the samples were stored at -20 oC. The digested products were verified with 1 % agarose gel.
|
m 1 2 3 4
|
1: pPG172 2: pPG172 3: pPG172 + SacI 4: pPG172 + SacII
|
Find the sequence of dacA in L.monocytogenes
Find the sequence of dacA in L.monocytogenes
We used the nucleotide sequence of ybbP from Bacillus subtilis to find its homology in Listeria monocytogenes. For the homology analysis, we used a web server called microbes online. It can be accessed using the following link: http://www.microbesonline.org/. We found a conserved hypothetical protein with 65.44 % similarity containing 273 amino acids (822 nucleotides) in L. monocytogenes (dacA). We also found that the cyclase domain (CD) sequence was present in the last half of the ybbP gene.
|
Type |
Sequences |
|
Protein |
>VIMSS7729571 (273 aa) MDFSNMSILHYLANIVDILVVWFVIYKVIMLIRGTKAVQLLKGIFIIIAVKLLSGFFGLQ TVEWITDQMLTWGFLAIIIIFQPELRRALETLGRGNIFTRYGSRIEREQHHLIESIEKST QYMAKRRIGALISVARDTGMDDYIETGIPLNAKISSQLLINIFIPNTPLHDGAVIIKGNE IASAASYLPLSDSPFLSKELGTRHRAALGISEVTDSITIVVSEETGGISLTKGGELFRDV SEEELHKILLKELVTVTAKKPSIFSKWKGGKSE |
|
CDS |
>VIMSS7729571 (822 nt)ATGGATTTTTCCAATATGTCGATATTGCATTATCTAGCAAATATTGTAGATATTCTTGTCGTATGGTTTGTAATTTATAAAGTGATCATGTTAATCCGAGGTACAAAAGCAGTACAATTATTAAAAGGCATTTTTATTATCATTGCAGTCAAACTATTAAGCGGATTTTTTGGTCTCCAAACAGTAGAATGGATTACGGATCAGATGCTTACTTGGGGATTCCTTGCAATTATAATTATCTTCCAACCGGAATTACGCCGTGCTTTAGAAACGCTTGGACGAGGCAATATTTTTACTCGTTATGGATCAAGAATAGAGCGTGAACAGCATCATTTAATCGAGTCTATCGAAAAGTCTACCCAGTATATGGCAAAACGTCGAATTGGGGCGCTGATTTCAGTGGCGCGCGATACAGGCATGGACGATTATATTGAAACAGGGATTCCGTTAAATGCAAAAATTTCTTCTCAATTATTAATTAATATTTTTATTCCGAATACACCGCTTCATGATGGAGCAGTTATTATTAAAGGAAACGAAATTGCATCGGCAGCAAGTTACTTGCCACTTTCAGATAGCCCGTTCTTATCCAAAGAACTTGGAACGCGTCACCGGGCTGCACTTGGGATTAGTGAAGTGACAGATAGTATTACGATTGTAGTTTCTGAAGAGACTGGCGGAATTTCCCTAACTAAAGGTGGAGAACTTTTCCGTGATGTGTCAGAAGAAGAGTTACATAAAATTCTTCTTAAAGAACTAGTCACAGTAACTGCAAAGAAACCTTCTATCTTTTCTAAATGGAAAGGAGGCAAAAGCGAATGA |
|
CDS +Flanking |
>VIMSS7729571 (250 nt upstream, 822 nt, 250 downstream)gatcgtgcagtcaaaatcgaagtttcaaataacggaacagtacggggtaatattacgaatatgggtattaaaaaaatctatggtattgttttgtaataaattaaaattaaagagcgctgaatgatttctttaacaaatctttccaatttttggcgggacgattgctttttccacctgcactcttatgctataataaagaatgtgaaccatgtgatcataaatggttttcaagaggcacggaggtgaagtgATGGATTTTTCCAATATGTCGATATTGCATTATCTAGCAAATATTGTAGA TATTCTTGTCGTATGGTTTGTAATTTATAAAGTGATCATGTTAATCCGAGGTACAAAAGCAGTACAATTATTAAAAGGCATTTTTATTATCATTGCAGTCAAACTATTAAGCGGATTTTTTGGTCTCCAAACAGTAGAATGGATTACGGATCAGATGCTTACTTGGGGATTCCTTGCAATTATAATTATCTTCCAACCGGAATTACGCCGTGCTTTAGAAACGCTTGGACGAGGCAATATTTTTACTCGTTATGGATCAAGAATAGAGCGTGAACAGCATCATTTAATCGAGTCTATCGAAAAGTCTACCCAGTATATGGCAAAACGTCGAATTGGGGCGCTGATTTCAGTGGCGCGCGATACAGGCATGGACGATTATATTGAAACAGGGATTCCGTTAAATGCAAAAATTTCTTCTCAATTATTAATTAATATTTTTATTCCGAATACACCGCTTCATGATGGAGCAGTTATTATTAAAGGAAACGAAATTGCATCGGCAGCAAGTTACTTGCCACTTTCAGATAGCCCGTTCTTATCCAAAGAACTTGGAACGCGTCACCGGGCTGCACTTGGGATTAGTGAAGTGACAGATAGTATTACGATTGTAGTTTCTGAAGAGACTGGCGGAATTTCCCTAACTAAAGGTGGAGAACTTTTCCGTGATGTGTCAGAAGAAGAGTTACATAAAATTCTTCTTAAAGAACTAGTCACAGTAACTGCAAAGAAACCTTCTATCTTTTCTAAATGGAAAGGAGGCAAAAGCGAATGAtggatcga attttaaataacaaatggtcgattcggattgtagccttactactcgcagccatcctttttacatcagttaatgcaaataataataacgccacgactttttcaacgacttcttctagtgattcagaagtcatcgagaatgtcccagtcaaagtatattatgataaaacgaatttatatatttcgggtattccagaaactgttacagtcacgctttcaggccctcgtagcatcgttcagtctgc |
