Team:Goettingen/NoteBook w10

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Revision as of 11:14, 18 September 2013

August

08th

Preparation of CFP and YFP Samples from Katrins Miniprep yesterday for sequencing by SeqLab, First Round RD of the parts for new cloning strategy, Colony PCR for Trasnformations from 6.8.13, Transformation from 6.8.13, Primer design for DarRrev for DarR reporter system

Preparation of CFP and YFP Samples from Katrins Miniprep yesterday for sequencing by SeqLab

Pipetted ~700ng of DNA + 0.25µl undiluted primer + Water to 7µl as follows:

1: CFP C1 VF2

2: CFP C2 VR

3: YFP C2 VF2

4: YFP C1 VR

Gel doc of PCR from Katrin yesterday test PCR part7 C1

M/2/4/8/NC

bands are ok so PCR purification was performed

-> marked as rev. terminator (in to do box)

newly prepared competent cells from yesterday (XL1 Blue) are tested with a 1 ng/ µl pbluescript Trafo and a trafo with just water and one without (see journal above)

put to 37°C at 13:00

inoculation of GFP positive cells

inoculation of part 8 and part1 from cryo stocks

First Round RD of the parts for new cloning strategy

Parts 1-4: PstI FD

YFP, CFP and Part 8: EcoRI FD

Katrin Gunkas Überprotokoll:

1.5µg DNA

4µl Enzyme

4µl Buffer FD

xµl H2O

=40µl reaction

-->incubation at 37°C for 1.5h

-->cleanup using the PCR cleanup kit, elution in 30+10µl (40 overall) prewarmed H2O

-->Nanodrop results on eppis (all around 30ng), about 1.2µg DNA retrieved in all samples (Which is highly unlikely and once again should make us think about nanodrop measurements)

Tomorrow 2nd round! Parts 1-4 with SpeI and the others with XbaI!

Transformation from 6.8.13

- plates: no colonies on 50 µl-plates observed, but on rest-plates

- Neg. control plates: no colonies

- w/o DarR insert plate: few colonies

- w/o Riboswitch insert plate: few colonies

- w/o Promoter3operator insert plate: few colonies

- Promoter3operator + pSB1C3: few colonies

- Riboswitch 40/41 + pSB1C3: several colonies

- Riboswitch 40/43 + pSB1C3: several colonies

- Riboswitch 41/42 + pSB1C3: several colonies

- Riboswitch 42/43 + pSB1C3: many colonies

- DarR + pSB1C3: many colonies

- Promoter3operator + part 6: some colonies --> see above (fluorescence microscopy)

- Plates stored in big fridge at 4 °C

Colony PCR for Trasnformations from 6.8.13

- according to protocol from ….; preparation of MasterMix for 55 reactions --> 25 µl

- PCR tube Colour/Number coding of clones:

o w/o DarR insert plate: 1 colony picked (A; white)

o w/o Riboswitch insert plate: 1 colony picked (B; white)

o w/o Promoter3operator insert plate: 1 colony picked (C; white)

o Promoter3operator + pSB1C3: 4 colonies picked (1, 2, 3, 4; yellow)

o Riboswitch 40/41 + pSB1C3: 8 colonies (5, 6, 7, 8, 9, 10, 11, 12; orange)

o Riboswitch 40/43 + pSB1C3: 9 colonies (13, 14, 15, 16, 17, 18, 19, 20, 21; pink)

o Riboswitch 41/42 + pSB1C3: 7 colonies (22, 23, 24, 25, 26, 27, 28; violet)

o Riboswitch 42/43 + pSB1C3: 10 colonies (29, 30, 31, 32, 33, 34, 35, 36, 37, 38; blue)

o DarR + pSB1C3: 10 colonies, but only first 8 inoculated for MiniPrep (not enough medium prepared for 48 and 47); (39, 40, 41, 42, 43, 44, 45, 46, 47, 48; green)

- PCR taken out, addition of 5 µl 5xLD to each reaction; reactions stored at 4 °C ON --> gel run tomorrow

- Inoculation of clones 48 and 47 in 4 ml LBCm and incubation ON at 37 °C, ca. 200 - 210 rpm

Primer design for DarRrev for DarR reporter system

- Sequence of DarR fwd primer + suffix with overhang

- Sequence of DarR rev primer + prefix with overhang

-> Ordered by Katrin G.

Fold ↑

07th

PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration, Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones, Primers for generating reverse terminator insert

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

- Primers iGEM_67/68/69/70

1x reaction (50 µl)

Component	Volume Negative control	Volume 1x primer concentration	Volume 2x primer concentration	Volume 4x primer concentration
5x HF buffer	10 µl	10 µl	10 µl	10 µl
dNTP mix (12.5 mM each)	2 µl	2 µl	2 µl	2 µl
Primer fwd (5 pmol)	2 µl	2 µl	4 µl	8 µl
Primer rev (5 pmol)	2 µl	2 µl	4 µl	8 µl
Chromosomal DNA B. subtilis 168 (or water for neg. control)	1 µl	1 µl	1 µl	1 µl
PfuS	1 µl	1 µl	1 µl	1 µl
dH2O	32 µl (12 µl)	32 µl (12 µl)	28 µl (8 µl)	20 µl (0 µl)

- preparation of master mix for 20 reactions containing

Component	Volume
dNTP mix (12.5 mM each)	40 µl
5x HF buffer	200 µl
PfuS	20 µl
dH2O	400 µl
Total	660 µl

- 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

Step	Temperature	Time
Initial denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	Different; see table below… (TA = TM (≈66 °C) – 6 °C)	35 s
Elongation	72 °C	2 min
Final elongation	72 °C	10 min
Hold	15 °C	∞

	Primers	TA = 0.5 * [Tm(primer1) + Tm(Primer2)] – 6 °C	insert size (no overhangs from primers)	Name of Protocol in cycler	Colour code of epi tube
Riboswitch with native Promoter and RBS	iGEM_69 iGEM_70	51 °C	466 bp	Ribo1	Orange
Riboswitch with native Promoter	iGEM_69 iGEM_68	55.2°C	370 bp	Ribo2	Violett
Riboswitch only	iGEM_67 iGEM_68	59.7°C	213 bp	Ribo3	Yellow
Riboswitch with native RBS	iGEM_70 iGEM_67	55.5 °C	403 bp	Ribo4	Blue

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	Primer amount (1:20 dilution)
Riboswitch with native Promoter and RBS	iGEM_69 iGEM_70	2 µl
Riboswitch with native Promoter	iGEM_69 iGEM_68	4 µl
Riboswitch only	iGEM_67 iGEM_68	4 µl
Riboswitch with native RBS	iGEM_70 iGEM_67	4 µl

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

Component	Volume Negative control	Volume 1x primer concentration	Volume 2x primer concentration	Volume 4x primer concentration
5x HF buffer	10 µl	10 µl	10 µl	10 µl
dNTP mix (12.5 mM each)	2 µl	2 µl	2 µl	2 µl
Primer fwd (5 pmol)	2 µl	2 µl	4 µl	8 µl
Primer rev (5 pmol)	2 µl	2 µl	4 µl	8 µl
Part 7 C1 plasmid (ca. 12 ng/µl) or water for negative control	1 µl	1 µl	1 µl	1 µl
PfuS	1 µl	1 µl	1 µl	1 µl
dH2O	32 µl (12 µl)	32 µl (12 µl)	28 µl (8 µl)	20 µl (0 µl)

- preparation of master mix for 5 reactions containing

Component	Volume
dNTP mix (12.5 mM each)	10 µl
5x HF buffer	50 µl
PfuS	5 µl
dH2O	100 µl
Total	165 µl

- 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

Step	Temperature	Time
Initial denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	53.5 °C	35 s
Elongation	72 °C	2 min
Final elongation	72 °C	10 min
Hold	15 °C	∞

-> run ON

Fold ↑

06th

Restriction digest of DarR (polyadenylated PCR product), Purification of pBluescript(RD with EcoRI) from gel, Ligation of DarR/hybridization oligo inserts with pSB1C3/part6, Gel run of DarR insert and pBluescript+EcoRI, Design of oligos for self-hybridization, Transformation of parts from distribution kit 2013 (CFP, YFP)

Restriction digest of DarR (polyadenylated PCR product)

-> Volume: 12 µl of unpurified DarR polyA reaction

- Preparation of the digest:

DarR polyA reaction mix	12 µl
EcoRI FD	1.5 µl
PstI FD	1.5 µl
10xFD buffer	2 µl
dH₂O	3 µl
Total	20 µl

-> 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)

Sample	ng/µl	A260nm/A280nm	A260nm/A230nm
DarR insert E+P	6.1	1.34	1.15

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)

Sample	ng/µl	A260nm/A280nm	A260nm/A230nm
pBluescript + EcoRI	6.6	1.92	0.09

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

Component	DarR	Hyb oligo + pSB1C3	Hyb oligo + part 6
Insert/dH₂O (w/o insert control)	8 µl (6.1 ng/µl)	8.05 µl	8.05 µl
T4 Ligase (ThermoScientific)	2 µl	2 µl	2 µl
T4 ligation buffer 10x	2 µl	2 µl	2 µl
Vector	1 µl (56.6 ng/µl)	5 µl (6.3 ng/µl)	3 µl (9.8 ng/µl)
dH₂O	7 µl	4.95 µl	2.95 µl
Total	20 µl	20 µl	20 µl

-> 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

Fold ↑

@@ Line 80: / Line 80: @@
 <div class="monat">August</div>
+<div class="tlob" id="tl_0808">
+<span class="date">08th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
+<div class="cont">
+<p class="timeline-title">Preparation of CFP and YFP Samples from Katrins Miniprep yesterday for sequencing by SeqLab, First Round RD of the parts for new cloning strategy, Colony PCR for Trasnformations from 6.8.13, Transformation from 6.8.13, Primer design for DarRrev for DarR reporter system</p>
+<div class="timeline-cont">
+<p class="c0"><span class="c8">Preparation of CFP and YFP Samples from Katrins Miniprep yesterday for sequencing by SeqLab</span></p><p class="c0"><span class="c8">&nbsp;</span><span>Pipetted ~700ng of DNA + 0.25µl undiluted primer + Water to 7µl as follows:</span></p><p class="c0"><span>&nbsp;1: CFP C1 VF2</span></p><p class="c0"><span>2: CFP C2 VR</span></p><p class="c0"><span>3: YFP C2 VF2</span></p><p class="c0"><span>4: YFP C1 VR</span></p><p class="c0 c13"><span></span></p><p class="c0"><span class="c8">Gel doc of PCR from Katrin yesterday test PCR part7 C1</span></p><p class="c0"><span class="c8">M/2/4/8/NC</span></p><img src="https://static.igem.org/mediawiki/2013/b/b4/Goe-08.08.13_RT-1.png" /><p class="c0"><span>bands are ok so PCR purification was performed</span></p><p class="c6 c16 c45"><span>-></span><span>&nbsp;</span><span>marked as rev. terminator (in to do box)</span></p><p class="c6 c13"><span></span></p><p class="c6"><span class="c8">newly prepared competent cells from yesterday (XL1 Blue) are tested with a 1 ng/</span><span>&nbsp;</span><span class="c8">µl pbluescript Trafo and a trafo with just water and one without (see journal above)</span></p><p class="c6"><span>put to 37°C at 13:00</span></p><p class="c6 c13"><span></span></p><p class="c6"><span class="c8">inoculation of GFP positive cells</span></p><p class="c6"><span class="c8">inoculation of part 8 and part1 from cryo stocks</span></p><p class="c6 c13"><span class="c8"></span></p><p class="c6"><span class="c8">First Round RD of the parts for new cloning strategy</span></p><p class="c6"><span>Parts 1-4: PstI FD</span></p><p class="c6"><span>YFP, CFP and Part 8: EcoRI FD</span></p><p class="c6"><span>&nbsp;</span></p><p class="c6"><span>Katrin Gunkas Überprotokoll:</span></p><p class="c6"><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.5µg DNA</span></p><p class="c6"><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4µl Enzyme</span></p><p class="c6"><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4µl Buffer FD</span></p><p class="c6"><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;xµl H</span><span class="c12">2</span><span>O</span></p><p class="c6"><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;=40µl reaction</span></p><p class="c6"><span>-->incubation at 37°C for 1.5h</span></p><p class="c6"><span>-->cleanup using the PCR cleanup kit, elution in 30+10µl (40 overall) prewarmed H2O</span></p><p class="c6"><span>-->Nanodrop results on eppis (all around 30ng), about 1.2µg DNA retrieved in all samples (Which is highly unlikely and once again should make us think about nanodrop measurements)</span></p><p class="c6"><span>Tomorrow 2</span><span class="c49">nd</span><span>&nbsp;round! Parts 1-4 with SpeI and the others with XbaI!</span></p><p class="c0 c7 c13"><span></span></p><p class="c0 c7"><span class="c8">Transformation from 6.8.13</span></p><p class="c0 c7"><span>-</span><span>&nbsp; &nbsp;</span><span>plates: no colonies on 50 µl-plates observed, but on rest-plates</span></p><p class="c0 c7"><span>- &nbsp;Neg. control plates: no colonies</span></p><p class="c0 c7"><span>- &nbsp;w/o DarR insert plate: few colonies</span></p><p class="c0 c7"><span>- &nbsp;w/o Riboswitch insert plate: few colonies</span></p><p class="c0 c7"><span>- &nbsp;w/o Promoter3operator insert plate: few colonies</span></p><p class="c0 c7"><span>- &nbsp;Promoter3operator + pSB1C3: few colonies</span></p><p class="c0 c7"><span>- &nbsp;Riboswitch 40/41 + pSB1C3: several colonies</span></p><p class="c0 c7"><span>- &nbsp;Riboswitch 40/43 + pSB1C3: several colonies</span></p><p class="c0 c7"><span>- &nbsp;Riboswitch 41/42 + pSB1C3: several colonies</span></p><p class="c0 c7"><span>- &nbsp;Riboswitch 42/43 + pSB1C3: many colonies</span></p><p class="c0 c7"><span>- &nbsp;DarR + pSB1C3: many colonies</span></p><p class="c0 c7"><span>- &nbsp;Promoter3operator + part 6: some colonies --> see above (fluorescence microscopy)</span></p><p class="c0 c7"><span>- &nbsp;Plates stored in big fridge at 4 °C</span></p><p class="c0 c7"><span>&nbsp;</span></p><p class="c0 c7"><span class="c8">Colony PCR for Trasnformations from 6.8.13</span></p><p class="c0 c7"><span>- &nbsp;according to protocol from ….; preparation of MasterMix for 55 reactions --> 25 µl</span></p><p class="c0 c7"><span>- &nbsp;PCR tube Colour/Number coding of clones:</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>w/o DarR insert plate: 1 colony picked (A; white)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>w/o Riboswitch insert plate: 1 colony picked (B; white)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>w/o Promoter3operator insert plate: 1 colony picked (C; white)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>Promoter3operator + pSB1C3: 4 colonies picked (1, 2, 3, 4; yellow)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>Riboswitch 40/41 + pSB1C3: 8 colonies (5, 6, 7, 8, 9, 10, 11, 12; orange)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>Riboswitch 40/43 + pSB1C3: 9 colonies (13, 14, 15, 16, 17, 18, 19, 20, 21; pink)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>Riboswitch 41/42 + pSB1C3: 7 colonies (22, 23, 24, 25, 26, 27, 28; violet)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; &nbsp;</span><span>Riboswitch 42/43 + pSB1C3: 10 colonies (29, 30, 31, 32, 33, 34, 35, 36, 37, 38; blue)</span></p><p class="c6 c23"><span>o</span><span>&nbsp; </span><span>DarR + pSB1C3: 10 colonies, but only first 8 inoculated for MiniPrep (not enough medium prepared for 48 and 47); (39, 40, 41, 42, 43, 44, 45, 46, 47, 48; green)</span></p><p class="c0 c7"><span>- &nbsp;PCR taken out, addition of 5 µl 5xLD to each reaction; reactions stored at 4 °C ON --> gel run tomorrow</span></p><p class="c0 c7"><span>- &nbsp;Inoculation of clones 48 and 47 in 4 ml LB</span><span class="c49">Cm</span><span>&nbsp;and incubation ON at 37 °C, ca. 200 - 210 rpm</span></p><p class="c0 c7 c13"><span></span></p><p class="c0 c7"><span class="c8">Primer design for DarR</span><span class="c8 c12">rev </span><span class="c8">for DarR reporter system</span></p><p class="c0 c7"><span>- &nbsp;Sequence of DarR fwd primer + suffix with overhang</span></p><p class="c0 c7"><span>- &nbsp;Sequence of DarR rev primer + prefix with overhang</span></p><p class="c6 c23"><span>-></span><span>&nbsp;</span><span>Ordered by Katrin G.</span></p><p class="c6 c13"><span></span></p>
+<div class="fbutton">Fold ↑</div>
+</div>
+</div>
+</div>
 <div class="tlob" id="tl_0807">
@@ Line 86: / Line 98: @@
 <p class="timeline-title">PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration, Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones, Primers for generating reverse terminator insert</p>
 <div class="timeline-cont">
 <p class="c0"><span class="c8">PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration</span></p><p class="c0"><span>-</span><span>&nbsp; &nbsp;</span><span>Primers iGEM_67/68/69/70</span></p><img src="https://static.igem.org/mediawiki/2013/c/c2/Goe-07.08.13-RT-1.png" /><p class="c0 c13"><span></span></p><p class="c0"><span>1x reaction (50 µl)</span></p><a href="#" name="030d6dd38fce9bb0601796cbd3cd5e216930e7fd"></a><a href="#" name="4"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c42"><p class="c0"><span class="c8">Component</span></p></td><td class="c31"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">Negative control</span></p></td><td class="c11"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">1x primer concentration</span></p></td><td class="c11"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">2x primer concentration</span></p></td><td class="c11"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">4x primer concentration</span></p></td></tr><tr><td class="c42"><p class="c0"><span>5x HF buffer</span></p></td><td class="c31"><p class="c0 c2"><span>10 µl</span></p></td><td class="c11"><p class="c0 c2"><span>10 µl</span></p></td><td class="c11"><p class="c0 c2"><span>10 µl</span></p></td><td class="c11"><p class="c0 c2"><span>10 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>dNTP mix (12.5 mM each)</span></p></td><td class="c31"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>Primer fwd (5 pmol)</span></p></td><td class="c31"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>4 µl</span></p></td><td class="c11"><p class="c0 c2"><span>8 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>Primer rev (5 pmol)</span></p></td><td class="c31"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>4 µl</span></p></td><td class="c11"><p class="c0 c2"><span>8 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>Chromosomal DNA </span><span class="c53">B. subtilis </span><span>168 (or water for neg. control)</span></p></td><td class="c31"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>PfuS</span></p></td><td class="c31"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>dH</span><span class="c12">2</span><span>O</span></p></td><td class="c31"><p class="c0 c2"><span>32 µl</span></p><p class="c0 c2"><span>(12 µl)</span></p></td><td class="c11"><p class="c0 c2"><span>32 µl</span></p><p class="c0 c2"><span>(12 µl)</span></p></td><td class="c11"><p class="c0 c2"><span>28 µl</span></p><p class="c0 c2"><span>(8 µl)</span></p></td><td class="c11"><p class="c0 c2"><span>20 µl</span></p><p class="c0 c2"><span>(0 µl)</span></p></td></tr></tbody></table><p class="c0"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;-</span><span>&nbsp;</span><span>preparation of master mix for 20 reactions containing</span></p><p class="c9 c16"><span>&nbsp;</span></p><a href="#" name="e3de1965059bee3dd5e42caf2416843ecb0ec180"></a><a href="#" name="5"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c28"><p class="c5"><span class="c8">Component</span></p></td><td class="c26"><p class="c5 c2"><span class="c8">Volume</span></p></td></tr><tr><td class="c28"><p class="c5"><span>dNTP mix (12.5 mM each)</span></p></td><td class="c26"><p class="c5 c2"><span>40 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>5x HF buffer</span></p></td><td class="c26"><p class="c5 c2"><span>200 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>PfuS</span></p></td><td class="c26"><p class="c5 c2"><span>20 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>dH</span><span class="c12">2</span><span>O</span></p></td><td class="c26"><p class="c2 c5"><span>400 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>Total</span></p></td><td class="c26"><p class="c5 c2"><span>660 µl</span></p></td></tr></tbody></table><p class="c0"><span class="c8">&nbsp;</span></p><p class="c6 c16 c33"><span>-</span><span>&nbsp;</span><span>addition of template (chrom. DNA/dH</span><span class="c12">2</span><span>O), 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</span></p><p class="c0"><span class="c8">&nbsp;</span></p><a href="#" name="4cf38c3210b742cfc68c876d35018e838ba55ba2"></a><a href="#" name="6"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c41"><p class="c9 c2"><span class="c8">Step</span></p></td><td class="c4"><p class="c9 c2"><span class="c8">Temperature</span></p></td><td class="c46"><p class="c9 c2"><span class="c8">Time</span></p></td></tr><tr><td class="c41"><p class="c9 c2"><span>Initial denaturation</span></p></td><td class="c4"><p class="c9 c2"><span>98.5 °C</span></p></td><td class="c46"><p class="c9 c2"><span>5 min</span></p></td></tr><tr><td class="c41"><p class="c9 c2"><span>Denaturation</span></p></td><td class="c4"><p class="c9 c2"><span>98.5 °C</span></p></td><td class="c46"><p class="c9 c2"><span>30 s</span></p></td></tr><tr><td class="c41"><p class="c9 c2"><span>Annealing</span></p></td><td class="c4"><p class="c9 c2"><span>Different; see table below…</span></p><p class="c9 c2"><span>(T</span><span class="c12">A</span><span>&nbsp;= T</span><span class="c12">M</span><span>&nbsp;(≈66 °C) – 6 °C)</span></p></td><td class="c46"><p class="c9 c2"><span>35 s</span></p></td></tr><tr><td class="c41"><p class="c9 c2"><span>Elongation</span></p></td><td class="c4"><p class="c9 c2"><span>72 °C</span></p></td><td class="c46"><p class="c9 c2"><span>2 min</span></p></td></tr><tr><td class="c41"><p class="c9 c2"><span>Final elongation</span></p></td><td class="c4"><p class="c9 c2"><span>72 °C</span></p></td><td class="c46"><p class="c9 c2"><span>10 min</span></p></td></tr><tr><td class="c41"><p class="c9 c2"><span>Hold</span></p></td><td class="c4"><p class="c9 c2"><span>15 °C</span></p></td><td class="c46"><p class="c9 c2"><span>∞</span></p></td></tr></tbody></table><p class="c0"><span class="c8">&nbsp;</span></p><p class="c0"><span class="c8">&nbsp;</span></p><a href="#" name="06d92520c2bdcbe14fe6f3484d428e7daef0bf2f"></a><a href="#" name="7"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c22"><p class="c0"><span class="c8">&nbsp;</span></p></td><td class="c30"><p class="c0"><span class="c8">Primers</span></p></td><td class="c60"><p class="c0"><span class="c8">T</span><span class="c12 c8">A</span><span class="c8">&nbsp;= 0.5 * [T</span><span class="c12 c8">m</span><span class="c49 c8">(primer1)</span><span class="c8">&nbsp;+ T</span><span class="c12 c8">m</span><span class="c49 c8">(Primer2)</span><span class="c8">] – 6 °C</span></p></td><td class="c43"><p class="c0"><span class="c8">insert size (no overhangs from primers)</span></p></td><td class="c24"><p class="c0"><span class="c8">Name of Protocol in cycler</span></p></td><td class="c24"><p class="c0"><span class="c8">Colour code of epi tube</span></p></td></tr><tr><td class="c22"><p class="c0"><span class="c8">Riboswitch </span></p><p class="c0"><span class="c8">with native Promoter and RBS</span></p></td><td class="c30"><p class="c0 c2"><span>iGEM_69</span></p><p class="c0 c2"><span>iGEM_70</span></p></td><td class="c60"><p class="c0 c2"><span>51 °C</span></p><p class="c0 c2"><span>&nbsp;</span></p></td><td class="c43"><p class="c0 c2"><span>466 bp</span></p></td><td class="c24"><p class="c0 c2"><span>Ribo1</span></p></td><td class="c24"><p class="c0 c2"><span class="c67">Orange</span></p></td></tr><tr><td class="c22"><p class="c0"><span class="c8">Riboswitch with native Promoter</span></p></td><td class="c30"><p class="c0 c2"><span>iGEM_69</span></p><p class="c0 c2"><span>iGEM_68</span></p></td><td class="c60"><p class="c0 c2"><span>55.2°C</span></p></td><td class="c43"><p class="c0 c2"><span>370 bp</span></p></td><td class="c24"><p class="c0 c2"><span>Ribo2</span></p></td><td class="c24"><p class="c0 c2"><span class="c62">Violett</span></p></td></tr><tr><td class="c22"><p class="c0"><span class="c8">Riboswitch only</span></p></td><td class="c30"><p class="c0 c2"><span>iGEM_67</span></p><p class="c0 c2"><span>iGEM_68</span></p></td><td class="c60"><p class="c0 c2"><span>59.7°C</span></p></td><td class="c43"><p class="c0 c2"><span>213 bp</span></p></td><td class="c24"><p class="c0 c2"><span>Ribo3</span></p></td><td class="c24"><p class="c0 c2"><span class="c61">Yellow</span></p></td></tr><tr><td class="c22"><p class="c0"><span class="c8">Riboswitch with native RBS</span></p></td><td class="c30"><p class="c0 c2"><span>iGEM_70</span></p><p class="c0 c2"><span>iGEM_67</span></p></td><td class="c60"><p class="c0 c2"><span>55.5 °C</span></p></td><td class="c43"><p class="c0 c2"><span>403 bp</span></p></td><td class="c24"><p class="c0 c2"><span>Ribo4</span></p></td><td class="c24"><p class="c0 c2"><span class="c48">Blue</span></p></td></tr></tbody></table><p class="c0"><span>&nbsp;</span></p><p class="c0"><span class="c8">Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>1.5 % Agarose-1xTAE gel</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>4 µl PCR reaction + 1 µl 5x DNA Loading Dye</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>5 µl of Test RDs; 1 µl uncut plasmid + 1 µl 5x DNA Loading Dye + 3 µl dH₂O</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>3 µl 2 log ladder</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>Run at ca. 100 V in 1xTAE buffer</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>Staining in EtBr and destaining in water</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp; </span><span>UV detection</span></p><p class="c0"><span>Gel 1:</span></p><img src="https://static.igem.org/mediawiki/2013/c/cb/Goe-07.08.13-RT-2.png" /><p class="c0 c13"><span></span></p><p class="c0"><span>Gel 2:</span></p><img src="https://static.igem.org/mediawiki/2013/9/9f/Goe-07.08.13-RT-3.png" /><p class="c23 c9 c55"><span>-></span><span>&nbsp;</span><span>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</span></p><p class="c23 c9 c55"><span>-></span><span>&nbsp;</span><span>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:</span></p><p class="c0"><span>&nbsp;</span></p><a href="#" name="8deb70100d07cd4a7452989496c64c50d35053e5"></a><a href="#" name="8"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c52"><p class="c0 c2"><span class="c8">&nbsp;</span></p></td><td class="c44"><p class="c0 c2"><span class="c8">Primers</span></p></td><td class="c47"><p class="c0 c2"><span class="c8">Primer amount (1:20 dilution)</span></p></td></tr><tr><td class="c52"><p class="c0 c2"><span class="c8">Riboswitch with native Promoter and RBS</span></p></td><td class="c44"><p class="c0 c2"><span>iGEM_69</span></p><p class="c0 c2"><span>iGEM_70</span></p></td><td class="c47"><p class="c0 c2"><span>2 µl</span></p></td></tr><tr><td class="c52"><p class="c0 c2"><span class="c8">Riboswitch with native Promoter</span></p></td><td class="c44"><p class="c0 c2"><span>iGEM_69</span></p><p class="c0 c2"><span>iGEM_68</span></p></td><td class="c47"><p class="c0 c2"><span>4 µl</span></p></td></tr><tr><td class="c52"><p class="c0 c2"><span class="c8">Riboswitch only</span></p></td><td class="c44"><p class="c0 c2"><span>iGEM_67</span></p><p class="c0 c2"><span>iGEM_68</span></p></td><td class="c47"><p class="c0 c2"><span>4 µl</span></p></td></tr><tr><td class="c52"><p class="c0 c2"><span class="c8">Riboswitch with native RBS</span></p></td><td class="c44"><p class="c0 c2"><span>iGEM_70</span></p><p class="c0 c2"><span>iGEM_67</span></p></td><td class="c47"><p class="c0 c2"><span>4 µl</span></p></td></tr></tbody></table><p class="c0"><span>&nbsp;</span></p><p class="c0"><span class="c8">Primers for generating reverse terminator insert</span></p><p class="c0"><span>&nbsp; &nbsp; -></span><span>&nbsp;</span><span>Primers arrived: dissolved in HPLC water and diluted 1:20</span></p><p class="c23 c9 c55"><span>-></span><span>&nbsp;</span><span>Primers tend to form secondary structures --> Test PCR for optimal primer concentration</span></p><p class="c23 c9 c55"><span>-></span><span>&nbsp;</span><span class="c40 c68">Annealing temperature</span><span>: </span><span class="c8">T</span><span class="c12 c8">A</span><span class="c8">&nbsp;= 0.5 * [T</span><span class="c12 c8">m</span><span class="c49 c8">(iGEM_71)</span><span class="c8">&nbsp;+ T</span><span class="c12 c8">m</span><span class="c49 c8">(iGEM_72)</span><span class="c8">] – 6 °C = 0.5 *[60 °C + 59.1] – 6 °C =</span><span class="c3 c40">53.55 °C</span></p><p class="c0"><span class="c40">&nbsp;</span></p><p class="c0"><span class="c40">TEST PCR</span></p><p class="c0"><span>1x reaction (50 µl) using part 7 C1 plasmid as template</span></p><p class="c0"><span>Plasmid concentration when used as a template: 10 – 20 ng/ reaction</span></p><p class="c0"><span>Part 7 C1 purified on 11.7.13; concentration = 120.5 ng/µl --> 1:10 dilution in water --> ca. 12 ng/µl</span></p><a href="#" name="a43e88f9d4ca07edfdd3c97e075bb33877815525"></a><a href="#" name="9"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c42"><p class="c0"><span class="c8">Component</span></p></td><td class="c31"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">Negative control</span></p></td><td class="c11"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">1x primer concentration</span></p></td><td class="c11"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">2x primer concentration</span></p></td><td class="c11"><p class="c0 c2"><span class="c8">Volume</span></p><p class="c0 c2"><span class="c8">4x primer concentration</span></p></td></tr><tr><td class="c42"><p class="c0"><span>5x HF buffer</span></p></td><td class="c31"><p class="c0 c2"><span>10 µl</span></p></td><td class="c11"><p class="c0 c2"><span>10 µl</span></p></td><td class="c11"><p class="c0 c2"><span>10 µl</span></p></td><td class="c11"><p class="c0 c2"><span>10 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>dNTP mix (12.5 mM each)</span></p></td><td class="c31"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>Primer fwd (5 pmol)</span></p></td><td class="c31"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>4 µl</span></p></td><td class="c11"><p class="c0 c2"><span>8 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>Primer rev (5 pmol)</span></p></td><td class="c31"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>2 µl</span></p></td><td class="c11"><p class="c0 c2"><span>4 µl</span></p></td><td class="c11"><p class="c0 c2"><span>8 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>Part 7 C1 plasmid (ca. 12 ng/µl) or water for negative control</span></p></td><td class="c31"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>PfuS</span></p></td><td class="c31"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td><td class="c11"><p class="c0 c2"><span>1 µl</span></p></td></tr><tr><td class="c42"><p class="c0"><span>dH</span><span class="c12">2</span><span>O</span></p></td><td class="c31"><p class="c0 c2"><span>32 µl</span></p><p class="c0 c2"><span>(12 µl)</span></p></td><td class="c11"><p class="c0 c2"><span>32 µl</span></p><p class="c0 c2"><span>(12 µl)</span></p></td><td class="c11"><p class="c0 c2"><span>28 µl</span></p><p class="c0 c2"><span>(8 µl)</span></p></td><td class="c11"><p class="c0 c2"><span>20 µl</span></p><p class="c0 c2"><span>(0 µl)</span></p></td></tr></tbody></table><p class="c0"><span>&nbsp;</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; </span><span>preparation of master mix for 5 reactions containing</span></p><p class="c9 c16"><span>&nbsp;</span></p><a href="#" name="469437c4b810888ff3b09287c6b6d7010a766572"></a><a href="#" name="10"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c28"><p class="c5"><span class="c8">Component</span></p></td><td class="c26"><p class="c5 c2"><span class="c8">Volume</span></p></td></tr><tr><td class="c28"><p class="c5"><span>dNTP mix (12.5 mM each)</span></p></td><td class="c26"><p class="c5 c2"><span>10 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>5x HF buffer</span></p></td><td class="c26"><p class="c5 c2"><span>50 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>PfuS</span></p></td><td class="c26"><p class="c5 c2"><span>5 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>dH</span><span class="c12">2</span><span>O</span></p></td><td class="c26"><p class="c5 c2"><span>100 µl</span></p></td></tr><tr><td class="c28"><p class="c5"><span>Total</span></p></td><td class="c26"><p class="c5 c2"><span>165 µl</span></p></td></tr></tbody></table><p class="c0"><span class="c8">&nbsp;</span></p><p class="c6 c16 c45"><span>-</span><span>&nbsp; &nbsp;</span><span>addition of template (chrom. DNA/dH</span><span class="c12">2</span><span>O), 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</span></p><p class="c9 c63"><span>&nbsp;</span></p><p class="c0"><span>PCR protocol</span></p><a href="#" name="c2edad18ba2ff10cfbae19a99a94c6921a817fd8"></a><a href="#" name="11"></a><table cellpadding="0" cellspacing="0" class="c21"><tbody><tr><td class="c11"><p class="c9 c2"><span class="c8">Step</span></p></td><td class="c19"><p class="c9 c2"><span class="c8">Temperature</span></p></td><td class="c56"><p class="c9 c2"><span class="c8">Time</span></p></td></tr><tr><td class="c11"><p class="c9 c2"><span>Initial denaturation</span></p></td><td class="c19"><p class="c9 c2"><span>98.5 °C</span></p></td><td class="c56"><p class="c9 c2"><span>5 min</span></p></td></tr><tr><td class="c11"><p class="c9 c2"><span>Denaturation</span></p></td><td class="c19"><p class="c9 c2"><span>98.5 °C</span></p></td><td class="c56"><p class="c9 c2"><span>30 s</span></p></td></tr><tr><td class="c11"><p class="c9 c2"><span>Annealing</span></p></td><td class="c19"><p class="c9 c2"><span>53.5 °C</span></p></td><td class="c56"><p class="c9 c2"><span>35 s</span></p></td></tr><tr><td class="c11"><p class="c9 c2"><span>Elongation</span></p></td><td class="c19"><p class="c9 c2"><span>72 °C</span></p></td><td class="c56"><p class="c9 c2"><span>2 min</span></p></td></tr><tr><td class="c11"><p class="c9 c2"><span>Final elongation</span></p></td><td class="c19"><p class="c9 c2"><span>72 °C</span></p></td><td class="c56"><p class="c9 c2"><span>10 min</span></p></td></tr><tr><td class="c11"><p class="c9 c2"><span>Hold</span></p></td><td class="c19"><p class="c9 c2"><span>15 °C</span></p></td><td class="c56"><p class="c9 c2"><span>∞</span></p></td></tr></tbody></table><p class="c0"><span>-></span><span>&nbsp;</span><span>run ON</span></p><p class="c0 c13"><span></span></p>