Team:Freiburg Bioware/NoteBook/Labjournal/October2

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Investigator: Achim, Hanna

Comment: Because yesterday's cloning didn't deliver a good separartion of the expected gel bands. Nevertheless ligation and trafo was performed. In order to immediately find out, whether we received successful results a colony PCR will be performed.
Two clones were picked from each plat. In addition to that a positive (pAAV_RC) and a negative control (pSB1C3_lITR) was prepared.
Used primer: 4200 rev and Cap3500 for. Expected fragment size: 885 bp.
PCR was performed following the standard protocol.

Freiburg10 1110 testdigestionhanna1label.png

Freiburg10 1110 testdigestionhanna2label.png

All samples match the positive control!
To do: Mini-Prep and sequencing.

Preparation of SDS-PAGE gel (10%)

Investigator: Hanna

Comment: In order to perform a Western Blot of different virus capsids (with and without capsid-motifs), 2 10% SDS polyacrylamid gels were prepared.

Resolving gel: 15 mL

H₂O: 5.9 mL
Acryl-bisacrylamide mix (30%): 5 mL
Tris (1.5 M, pH 8.8): 3.8 mL
SDS (10%): 0.15 mL
Ammonium persulfate (10%): 0.15 mL
TEMED: 0.006 mL

Stacking gel (5%): 5 mL

H₂O: 3.4 mL
Acryl-bisacrylamide mix (30%): 0.83 mL
Tris (1.5 M, pH 6.8): 0.63 mL
SDS (10%): 0.05 mL
Ammonium persulfate (10%): 0.05 mL
TEMED: 0.005 mL

Colony PCR of p40_VP123_capins

Investigator: Bea

Comment: Since the first attempt did not work,and no cells grew on the plate and the same ligation was transformed again into BL-21 and a lot of clones grew on the plate, I decided to perform a colony PCR in order to check several colonies and to inoculate at the same day for a Midi-Prep.

Protocol:

Primer used: O162
Primer used: O38

The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture:

Result: We can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.

Cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD

Investigator: Stefan

Comment: To produce another GOI for testing in cell culture, the cytosine deaminase needs to be assembled with lITR_promotor_betaglobin. In the next step hgH_rITR needs to be added.

Vector name:
pSB1C3_CD clone 1
pSB1C3_CD clone 2

Insert name:
pSB1C3_lITR_CMV_beta-globin (P729)
pSB1C3_lITR_phTERT_beta-globin (P730)

Digestion:

components	volume CD clone 1 + 2 /µl	volume P729 /µl	volume P730 /µl
DNA	6	14	6
BSA (10x)	2	2	2
Buffer 4 (10x)	2	2	2
Enzyme EcoI	1	1	1
Enzyme XbaI	1	-	-
Enzyme SpeI	-	1	1
H2O	8	-	8
Total volume (e.g. 15,20,25,30 µl)	20	20	20

Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt

CD clone 1 yielded to bands around 2500 bp to 3000 bp. Since the vector was cut only using EcoRI and SpeI, it was expected to be linearized, not to be cut into two fragments this size. Therefore, this sample was discarded and cloning was continued using CD clone 2.

Gel extraction:
Was performed according to protocol.

T4 Ligation:

ligation name	729 + CD cl2	730 + CD cl2
volume of vector	3,67	2,82
volume of insert	4,33	5,18
T4 ligase buffer (10x)	1	1
T4 ligase	1	1

Transformation:
Was performed according to standard protocol using BL21 cells.

Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay

Investigator: Kerstin, Anissa

Seeding 4x 96-well plates: 2x HT1080 and 2x A431

FACS-Analysis

Investigator: Kerstin

...

Preparation of the ELISA

Investigator: Volker

The AAV particle standard that contains 3.6x10^9 viral particles was dissolved in 500µl as described in the protocol of the Progen AAV Titration ELISA and a absorption spectrum was measured. These purified and concentrated viral particles could be used for biophysical measurements, there for the possibility to detect the viral particles by absorption was interesting for us.

The Spectrum measured in the NanoDrop is the following:

Freiburg10 Absorption spectrum of the AAV particle standard.jpeg

Trafo evaluation of Rep52

Investigator:Kira
T4 ligation seems to worked out. The plate contained many colonies.

147. labday 12.10.2010

Sequencing analysis: pSB1C3_mGMK_TK30 and pSB1C3_CD

Investigator: Stefan

Test digestion of pSB1C3_CD and pSB1C3_CD_SDM-PstI_new

Investigator: Stefan

Comment: Nachricht von Stefan an Stefan: bitte ergänzen!

Comment: Sequencing results revealed that there is CFP rather than CD in P819. In order to verify this and exclude the possibility that the wrong plasmid was sent for sequencing test digestions were performed. Additionally the original vector at which the SDM to remove the PstI was performed was digested as well to verify its insert.

Components	digest 1 / µl	digest 2 / µl
DNA	1	7
Buffer 4	1	1
BSA (10x)	1	1
Enzyme I	SpeI 0,3	PstI 0,3
Enzyme II	XbaI 0,3	XbaI 0,3
H₂O	6,4	0,4
Total volume	10	10

Comment: Using these digestion approaches, it was expected for CFP to deliver always bands at ~750 bp. For the CD it was expected to show bands at 90 bp, 1200 bp and 2000 bp if the CD still contains the PstI restictiton site, 1300 bp and 2000 bp if it was removed.

Gel:
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes

Comment: As it can be seen, P819 does contain CFP, therefore a mistakenly sequenced plasmid can be excluded. Also it can be seen that P690 contains the CD. Therefore an additonal SDM of P690 to remove the PstI restriction site is necessary.

qPCR of virus stock: P816/ TKGMk-Standard; R/C: P468 from 5.10. (VLP harvested by Adrian)

Investigator: Achim

Protocol for quantitative real-time PCR of virus particles

Date: 12.10.

Achim

Virus Stock: A.F.; P816; TKGMK-Standard; R/C: P468; 5.10; consists of supernatant from pelleted cell fragments.

Following the Protocol used by (Rohr et al., 2002).

I digested 5 µl virus dilution with 7,5 µl DNAse I (7,5 units) and 25 µl 50 mM MgCl2 (end concentration 25 mM) in a final volume of 50 µl at 37°C for 30 min.

è DNAse should be heat inactivated at 65°C for 10 min. I forgot that step. The enzyme should however be inactivated in the initial denaturation step.

I prepared the following PCR reactions:

Sample	1	2	3	4	5	6	7	8		9	10
	1.1	1.2	2.1	2.2	S.1	S.2	NP1	NP2	MM for samples 1 -8	NH1	NH2
PCRmix	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	112.5	12.5	12.5
Primer for	1	1	1	1	1	1	1	1	9	-	-
Primer rev	1	1	1	1	1	1	1	1	9	-	-
Template	2	2	2	2	5	5	-	-	-	-	-
H2O	8.5	8.5	8.5	8.5	5.5	5.5	10.5	10.5	-	12.5	12.5
Total	25	25	25	25	25	25	25	25		25	25

Step	Time	Temp
Initial denaturation	7’	95°C

Denaturation	10’’	95°C
Annealing/Extension	30’’	60°C
Number of Cycles	45

Results:

Freiburg10 2010-10-12 qPCR Auswertung.PNG

è Both negative controls containing primers were contaminated with DNA.

è Negative controls containing only H2O were negative.

è Values for plasmids/reaction vary; from 1 – 100 for the same virus stock

è Dilution of the virus solution: 1:10; 2µl PCR sample: 3.2 * 10^5 virus particles/ml

è Because of the contaminated negative controls and the high deviation, I will repeat the experiment tomorrow to verify the results.

References:

Rohr, U., Wulf, M., Stahn, S., Steidl, U., Haas, R., Kronenwett, R., et al. (2002). Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR. Journal of virological methods, 106(1), 81-8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12367732.

Cloning sr39 into mGMK_TK30

Investigator: Bea

Comment: We do not only want to submit the tk30, a mutant version of the thymidine kinase, but as well another mutant protein which is named sr39. SR39 seems to have a better activity than the tk30 without being fused to the guanylate kinase (mgmk), but the sr39 also works quite well as a fusion protein. We ordered one part of the sr39 and we are now subcloning it into the mgmnk_tk30 construct in order to obtain mgmk_sr39.

Protocol:

Freiburg10 SR39 into mGMK tk30 12.10.PNG

The digested fragments were loaded on a 1.2% agarose gel. The results can be seen above in the gel picture: I cut out the small visible band in the right lane, and the upper intensive band of the left lane.

Result:

Freiburg10 Digest sr39 and mGMK tk30.png

After gel extraction has been performed, I ligated the two fragments and transformed BL-21 cells, plated them on cm agar plates and incubated them over night at 37°C.

Concentration of virus stock for Western Blot analysis

Investigator: Hanna

For the first Western Blot try, we investigate whether is is enough to concentrate the virus stock (from cell supernatant) and then to simply load it onto a SDS-PAGE gel and perfom immuno-staining.
For this purpose 8 mL virus stock (pHelper, pAAV_RC, GOI=YFP) were concentrated via amicon filtration.

First, amicon filter was washed with 4 mL PBS.
Then 4 mL virus stock was loaded onto the filter and centrifuged at 3000 rpm for 15 minutes.
Flow-through was discarded and additional virus stock was loaded, centrifuged at 3000 rpm for 25 minutes.
Flow-through was discarded, remaining solution was re-suspended and additional virus stock was added. Amicon filter was centrifuged at 3000 rpm for 50 minutes.
Flow-through was discarded. Residual protein solution: 500 µL. Filter was washed with 3 mL PBS, centrifuging 35 minutes two times.
2 x 40 µL protein solution (16x) was taken and mixed with Laemmli buffer and stored over night @ 4°C.

To do: SDS-PAGE and Western Blot tomorrow.
Perfomance of another try by harvesting viruses of the cell pellet with RIPA buffer & freeze/ thaw.
Western Blot of unmodified, and modified capsids (N-terminal fusion and VP1 insertion with CFP ref. mVenus) --> size shift should be detectable.

Transduction of HT1080 and A431

Investigator: Kerstin

Mini-Prep and test digestion of several constructs

Investigator: Stefan

Glycerol stocks were prepared:

B651 = pSB1C3_P40_VP123
B652 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR
B653 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR
B654 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR
B655 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR
B656 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO
B657 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO
B658 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO
B659 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO
B660 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR
B661 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR

Mini-Prep was performed according to standard protocol:

P821 = pSB1C3_P40_VP123 c = 327,4 ng/µl
P822 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 342,9 ng/µl
P823 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 324,0 ng/µl
P824 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 81,6 ng/µl
P825 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 148,4 ng/µl
P826 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 105,8 ng/µl
P827 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 121,1 ng/µl
P828 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 188,4 ng/µl
P829 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 140,4 ng/µl
P830 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR c = 197,2 ng/µl
P831 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR c = 207,3 ng/µl

Test digestion:

Components	P822 + P823 / µl	P824 + P825 / µl	P830 + P831 / µl
DNA	1,5	1,5	2
Buffer 4	1	1	1
BSA (10x)	1	1	1
PstI	0,3	-	-
XbaI	0,3	0,3	0,3
SpeI	-	0,3	0,3
H₂O	5,9	5,9	5,4
Total volume	10	10	10

Gel:
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes

Comment: P822 and P823 smear a lot, therefore evaluation is difficult. Another test digestion would be reasonable.
P824 contains hgH_rITR, therefore it will be discarded. P825 looks well and was sent for sequencing.
P830 and P831 look well and will be sent for sequencing tomorrow.

Repetiton: Site-directed mutagenesis in pSB1C3_CD construct

Investigator: Stefan

Comment: Sequencing revealed that there is no CD in our pSB1C3_CD but rather CFP. So, anonther approach of the SDM needs to be performed.

Protocol using the QuickChange Lightning Kit from Stratagene:

Ingredients:

Ingredients	Volume / µl
10x reaction buffer	2,5
dNTP	0,5
forward primer: O191	0,56
reverse primer: O192	0,57
DNA Template	0,5 (1:10 dilution)
QuikSolution Reagent	0,75
QuikChangeLightning Enzyme	0,5
H₂O	14,35
Total volume	25

The following PCR program was used:

95 °C 120 s
95 °C 20 s
60 °C 60 s
68 °C 105 s
Goto step 2 repeat 17 times
68 °C 300 s

Digestion using DpnI:
To digest methylated and hemi-methylated DNA, 1 µl of DpnI was added and the mixture was incubated for 10 minutes at 37 °C.
Transformation:
Trafo was performed according to protocol, but using XL1b cells instead of XL-10 Gold cells.

148. labday 13.10.2010

qPCRs of virus stocks

Investigator: Achim

Sample:	Plasmid, Glycerol stock:	Sample No.
pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 1	B561	1
pSB1C3_001_RC_IRCK_P5tataless clone1	P668	2
pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1	P378	3
pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1	P377	4
pAAV_RC_RepCapIns_SDMKpnI	P468	5

Digestion: 37°C, 30 min

	1	2	3	4	5	MM x6
Virus solution	5	5	5	5	5
DNAseI (7,5 U)	7,5	7,5	7,5	7,5	7,5	45
MgCl2 50 mM	5	5	5	5	5	30
H2O	32.5	32.5	32.5	32.5	32.5	195
Total	50	50	50	50	50	300

PCR reaction:

Sample	1	2	3	4	5	6	7	8	9	10	11	12	13	14		15	16
	1.1	1.2	2.1	2.2	3.1	3.2	4.1	4.2	5.1	5.2	S.1	S.2	NP1	NP2	MM for samples 1 -14 (x15)	NH1	NH2
PCRmix	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	12.5	187,5	12.5	12.5
Primer for 1:10	1	1	1	1	1	1	1	1	1	1	1	1	1	1	15	-	-
Primer rev 1:10	1	1	1	1	1	1	1	1	1	1	1	1	1	1	15	-	-
Template	2	2	2	2	2	2	2	2	2	2	5	5	-	-	-	-	-
H2O	8.5	8.5	8.5	8.5	8.5	8.5	8.5	8.5	8.5	8.5	5.5	5.5	10.5	10.5	-	12.5	12.5
Total	25	25	25	25	25	25	25	25	25	25	25	25	25	25		25	25

Team:Freiburg Bioware/NoteBook/Labjournal/October2

From 2010.igem.org

Revision as of 09:14, 14 October 2010

Contents

146. labday 11.10.2010

Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3

Preparation of SDS-PAGE gel (10%)

Colony PCR of p40_VP123_capins

Cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD

Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay

FACS-Analysis

Preparation of the ELISA

Trafo evaluation of Rep52

147. labday 12.10.2010

Sequencing analysis: pSB1C3_mGMK_TK30 and pSB1C3_CD

Test digestion of pSB1C3_CD and pSB1C3_CD_SDM-PstI_new

qPCR of virus stock: P816/ TKGMk-Standard; R/C: P468 from 5.10. (VLP harvested by Adrian)

Cloning sr39 into mGMK_TK30

Concentration of virus stock for Western Blot analysis

Transduction of HT1080 and A431

Mini-Prep and test digestion of several constructs

Repetiton: Site-directed mutagenesis in pSB1C3_CD construct

148. labday 13.10.2010

qPCRs of virus stocks

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+    szHTML+="<td height=1 width=1></td><td></td>";
+   }
+  }
+  szHTML+="</tr>";
+ }
+ szHTML+="</table></body></html>";
+ with (frames['frTabs'].document) {
+  open("text/html","replace");
+  charset=document.charset;
+  write(szHTML);
+  close();
+ }
+}
+function fnInit()
+{
+ g_rglTabX[0]=0;
+ var i;
+ for (i=1;i<=c_lTabs;i++)
+  with (frames['frTabs'].document.all.tbTabs.rows[1].cells[fnTabToCol(i-1)])
+   g_rglTabX[i]=offsetLeft+offsetWidth-6;
+}
+function fnTabToCol(iTab)
+{
+ return 2*iTab+1;
+}
+function fnNextTab(fDir)
+{
+ var iNextTab=-1;
+ var i;
+ with (frames['frTabs'].document.body) {
+  if (fDir==0) {
+   if (scrollLeft>0) {
+    for (i=0;i<c_lTabs&&g_rglTabX[i]<scrollLeft;i++);
+    if (i<c_lTabs)
+     iNextTab=i-1;
+   }
+  } else {
+   if (g_rglTabX[c_lTabs]+6>offsetWidth+scrollLeft) {
+    for (i=0;i<c_lTabs&&g_rglTabX[i]<=scrollLeft;i++);
+    if (i<c_lTabs)
+     iNextTab=i;
+   }
+  }
+ }
+ return iNextTab;
+}
+function fnScrollTabs(fDir)
+{
+ var iNextTab=fnNextTab(fDir);
+ if (iNextTab>=0) {
+  frames['frTabs'].scroll(g_rglTabX[iNextTab],0);
+  return true;
+ } else
+  return false;
+}
+function fnFastScrollTabs(fDir)
+{
+ if (c_lTabs>16)
+  frames['frTabs'].scroll(g_rglTabX[fDir?c_lTabs-1:0],0);
+ else
+  if (fnScrollTabs(fDir)>0) window.setTimeout("fnFastScrollTabs("+fDir+");",5);
+}
+function fnSetTabProps(iTab,fActive)
+{
+ var iCol=fnTabToCol(iTab);
+ var i;
+ if (iTab>=0) {
+  with (frames['frTabs'].document.all) {
+   with (tbTabs) {
+    for (i=0;i<=4;i++) {
+     with (rows[i]) {
+      if (i==0)
+       cells[iCol].style.background=c_rgszClr[fActive?0:2];
+      else if (i>0 && i<4) {
+       if (fActive) {
+        cells[iCol-1].style.background=c_rgszClr[2];
+        cells[iCol].style.background=c_rgszClr[0];
+        cells[iCol+1].style.background=c_rgszClr[2];
+       } else {
+        if (i==1) {
+         cells[iCol-1].style.background=c_rgszClr[2];
+         cells[iCol].style.background=c_rgszClr[1];
+         cells[iCol+1].style.background=c_rgszClr[2];
+        } else {
+         cells[iCol-1].style.background=c_rgszClr[4];
+         cells[iCol].style.background=c_rgszClr[(i==2)?2:4];
+         cells[iCol+1].style.background=c_rgszClr[4];
+        }
+       }
+      } else
+       cells[iCol].style.background=c_rgszClr[fActive?2:4];
+     }
+    }
+   }
+   with (aTab[iTab].style) {
+    cursor=(fActive?"default":"hand");
+    color=c_rgszClr[3];
+   }
+  }
+ }
+}
+function fnMouseOverScroll(iCtl)
+{
+ frames['frScroll'].document.all.tdScroll[iCtl].style.color=c_rgszClr[7];
+}
+function fnMouseOutScroll(iCtl)
+{
+ frames['frScroll'].document.all.tdScroll[iCtl].style.color=c_rgszClr[6];
+}
+function fnMouseOverTab(iTab)
+{
+ if (iTab!=g_iShCur) {
+  var iCol=fnTabToCol(iTab);
+  with (frames['frTabs'].document.all) {
+   tdTab[iTab].style.background=c_rgszClr[5];
+  }
+ }
+}
+function fnMouseOutTab(iTab)
+{
+ if (iTab>=0) {
+  var elFrom=frames['frTabs'].event.srcElement;
+  var elTo=frames['frTabs'].event.toElement;
+  if ((!elTo) ||
+   (elFrom.tagName==elTo.tagName) ||
+   (elTo.tagName=="A" && elTo.parentElement!=elFrom) ||
+   (elFrom.tagName=="A" && elFrom.parentElement!=elTo)) {
+   if (iTab!=g_iShCur) {
+    with (frames['frTabs'].document.all) {
+     tdTab[iTab].style.background=c_rgszClr[1];
+    }
+   }
+  }
+ }
+}
+function fnSetActiveSheet(iSh)
+{
+ if (iSh!=g_iShCur) {
+  fnSetTabProps(g_iShCur,false);
+  fnSetTabProps(iSh,true);
+  g_iShCur=iSh;
+ }
+}
+ window.g_iIEVer=fnGetIEVer();
+ if (window.g_iIEVer>=4)
+  fnBuildFrameset();
+//-->
+</script>
+<![endif]><!--[if gte mso 9]><xml>
+ <x:ExcelWorkbook>
+  <x:ExcelWorksheets>
+   <x:ExcelWorksheet>
+    <x:Name>Tabelle1</x:Name>
+    <x:WorksheetSource
+     HRef="2010-10-13%20qPCR%20auswertung_html-Dateien/sheet001.htm"/>
+   </x:ExcelWorksheet>
+   <x:ExcelWorksheet>
+    <x:Name>Tabelle2</x:Name>
+    <x:WorksheetSource
+     HRef="2010-10-13%20qPCR%20auswertung_html-Dateien/sheet002.htm"/>
+   </x:ExcelWorksheet>
+   <x:ExcelWorksheet>
+    <x:Name>Tabelle3</x:Name>
+    <x:WorksheetSource
+     HRef="2010-10-13%20qPCR%20auswertung_html-Dateien/sheet003.htm"/>
+   </x:ExcelWorksheet>
+  </x:ExcelWorksheets>
+  <x:Stylesheet
+   HRef="2010-10-13%20qPCR%20auswertung_html-Dateien/stylesheet.css"/>
+  <x:WindowHeight>12840</x:WindowHeight>
+  <x:WindowWidth>19440</x:WindowWidth>
+  <x:WindowTopX>120</x:WindowTopX>
+  <x:WindowTopY>60</x:WindowTopY>
+  <x:ProtectStructure>False</x:ProtectStructure>
+  <x:ProtectWindows>False</x:ProtectWindows>
+ </x:ExcelWorkbook>
+</xml><![endif]-->
+</head>
+<frameset rows="*,39" border=0 width=0 frameborder=no framespacing=0>
+ <frame src="2010-10-13%20qPCR%20auswertung_html-Dateien/sheet001.htm" name="frSheet">
+ <frame src="2010-10-13%20qPCR%20auswertung_html-Dateien/tabstrip.htm" name="frTabs" marginwidth=0 marginheight=0>
+ <noframes>
+  <body>
+   <p>Diese Seite verwendet Frames. Frames werden von Ihrem Browser aber nicht unterstützt.</p>
+  </body>
+ </noframes>
+</frameset>
 </html>