Team:Goettingen/NoteBook w3

Transformation from yesterday worked! 6 clones! And the Without-Insert-Control lacks clones!

è     Plates stored at 4 °C

Concentration of stored PCR products from 19.6.13

No. of PCR cycles	c(DNA) [ng/µl]	A260/A280	A260/A230
PCR product 10 cycles	1068.7	1.63	1.07
PCR product 20 cycles	975.7	1.66	1.10
PCR product 30 cycles	964.9	1.65	1.11

 

Digestion of PCR product (AND WHATS IN THE PCR?) (10 cycles) and Plasmid backbone (pSB1A3 (with AmpR))

According to: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones

 

Ezyme master mix:

5 µl Buffer 0

0.5 µl EcoRI

0.5 µl PstI

18 µl dH₂O

 

Digestion of backbone resp. insert:

4 µl linearized plasmid backbone (25 ng/µl for 100 ng total)

4 µl Enzyme master mix

resp.:

5 µl insert

4 µl Enzyme master mix

resp.:

10 µl insert

4 µl Enzyme master mix

 

digest 37°C/30 min heat kill 80°C/20 min

 

Ligation of backbone and insert:

According to: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones

 

2 µl digested plasmid backbone

10 µl EcoRI and PstI digested fragment

1.5 µl T4 DNA Ligase buffer

0.5 µl T4 DNA Ligase

1 µl H₂O

ligate 30°C/30 min heat kill 80°C/min

 

Transformation of ligated parts

According to method book

Digestion of amplified DAC-domains (S.aureus and S. pneumonia) from 17.06.

 

Components:	Volume
plasmid DNA	16µl
PstI	6µl
EcoRI	6µl
orange buffer (10x)	8µl
H2O	44µl

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

 

Digestion of prepared Strep-Tag from 17.06.

 

Components:	Volume
Strep-Tag	16µl
PstI	6µl
EcoRI	6µl
orange buffer (10x)	8µl
H2O	44µl

 

è incubation for 30min at 37°C

è heat inactivation for 20min at 65°C

è store at -20°C

 

è Preparation of Strap-Tag worked

è Preparation of DAC domain of S.aureus worked

è Preparation of DAC domain of S- pneumonia failed = EcoRI restriction site in fragment

 

PCR using primer iGEM 36,37 for construction of DarR operator biobrick.

Same recipe as yesterdays PCR, rest the protocol,

98.5	5min
98.5	30s	10 cycle	20cycle	30cycle
61.0	30s
72.0	2min
72.0	10min

 Ctrl: no enzyme

Increase the gel concentration to 4%.

PCR worked, Gel here:

Lane 1-8: ladder, iGEM36, iGEM37, ctrl(no Polyermase), 1 cycle, 10cycle, 20cycle, 30cycle.

Gel concentration : 4%

1cycle PCR product from yesterday.

So, use 4% gel, 10cycle PCR is enough

DarR sequencing results

in “our” genomic DNA of M. smegmatis, there are two additional codons inserted right after the GTG start codon:

“our” gDNA: GTG  AGC  GCG  AGC  GCC à Ser-Ala-Ser-Ala

Paper gDNA: GTG  _ _ _  _ _ _  AGC  GCC à Ser-Ala

Additionally, there are 5 point mutations:

120 C à G

132 A à G

210 T à C

228 C à T

263 T à C

All are silent, except for one: aa 87 Val à Ala (both small, hydrophobic aa)

Primer Design

Design of new DarR biobrick fwd primer (since 2 additional codons inserted and old primer designed for DarR gene without these codons)

Plate reader assay

-          Plate thrown away after ON measurement

-          Results:

 

Concentration of plasmid purified from Clone 2 of Part 2

Part no.	c(DNA) [ng/µl]	A260/A280	A260/A230
2 C2	120.6	1.87	2.24

 

Test restriction of Part 2 Clone 2

Component	Uncut control	SpeI single digestion	PstI single digestion	SpeI/PstI double digestion
Plasmid DNA (150 ng)	1.2 µl	1.2 µl	1.2 µl	1.2 µl
10x buffer	1 µl (Tango)	1 µl (Tango)	1 µl (buffer 0)	1 µl (Tango)
SpeI	-	1 µl	-	1 µl
PstI	-	-	1 µl	2 µl
dH2O	7.8 µl	6.8 µl	6.8 µl	4.8 µl
Total	10 µl	10 µl	10 µl	10 µl

-          Incubation for ca. 1.5 h at 37 °C (incubator)

-          Addition of 2 µl of 5x DNA loading dye and run on 1 % 1xTAE-agarose gel (marker: 100 bp ladder)

-          EtBr staining and destaining in H₂O, UV detection

è     Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

Conclusions:

è     Clone 2 contains the expected plasmid harboring part 2 (impure stock?)

 

Primer design and ordering

Design of primer iGEM_43

Ordering of primers iGEM_38 to iGEM_43 from Sigma-Aldrich

 

PCR:

PCR using primer iGEM32,33 for construction of DarR biobrick.

Because of those two primers have high tendency to form 2^nd structure, 1x 2x 4x primers are applied, to see the recipe, see dropbox > Reporter Team > PCR_iGEM32_33.xls

PCR was successful, better use 2x primer. Protocol as follows:

98.5	5min
98.5	30s	30 cycles
67.0	35s
72.0	2min
72.0	10min

 

è     Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

PCR using primer iGEM 36,37 for construction of DarR operator biobrick. To see the recipe, see dropbox > Reporter Team > PCR_iGEM32_33.xls

Protocol as follows:

98.5	5min	Only one cycle
98.5	30s
61.0	30s
72.0	12min

 

è     Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

 

2% gel cannot separate the primers and PCR product:

iGEM36:36nt

iGEM37:35nt

PCR product should be 57bp

Gel1: forget the primer as ctrl

Gel2: load the wrong primer