Team:Freiburg Bioware/NoteBook/Labjournal/October2

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146. labday 11.10.2010

Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3

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
Freiburg10 ColonyPCR p40vp123 1110.PNG


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.

Freiburg10 ColonyPCR p40VP123.png


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 6 146
BSA (10x) 2 22
Buffer 4 (10x)2 22
Enzyme EcoI11 1
Enzyme XbaI1- -
Enzyme SpeI-11
H2O8- 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



Freiburg10 cloning 111010.jpg


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 cl2730 + CD cl2
volume of vector 3,672,82
volume of insert4,335,18
T4 ligase buffer (10x)11
T4 ligase 11


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

Freiburg10 sequencingmGMK TK30 SDM PstI.jpg
Freiburg10 sequencing CD CFP.jpg


Test digestion of pSB1C3_CD and pSB1C3_CD_SDM-PstI_new

Investigator: Stefan


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

Freiburg10 test digestion 121010 1.jpg

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


Freiburg10 Transductionplan 11.10.2010.jpg
Freiburg10 Transductionplan1 11.10.2010.jpg
Freiburg10 Transductionplan2 11.10.2010.jpg
Freiburg10 Transductionplan3 11.10.2010.jpg
Freiburg10 Transductionplan4 11.10.2010.jpg

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

Freiburg10 test digestion 121010 2.jpg

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
dNTP0,5
forward primer: O191 0,56
reverse primer: O192 0,57
DNA Template0,5 (1:10 dilution)
QuikSolution Reagent0,75
QuikChangeLightning Enzyme0,5
H2O14,35
Total volume25



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

 

Results:

Freiburg10 13 10 auswertung.PNG

SDS Page of unmodified AAV2 capsids

Investigator: Hanna

Comment: Yesterday a 8x concentration of AAV2 supernatant-stock was performed. 10 µL Laemmli-buffer was added to 40 µL AAV stock (8x) and stored over night at 4°C.
Today a SDS-PAGE of these samples will be performed.

Samples were incubated for 10 minutes at 95°C for denaturation.

Loading plan:

5 µL PageRuler Prestained Protein Ladder 10 µL Sample 15 µL Sample 25 µL Sample


After running front reached the end of the gel, PAGE was stopped.

Western Blot:
Three layers of transfer buffer soaked whatman paper were placed onto the blotter. A polyvinylidene difluoride membrane was activated by methanol (5 seconds) and then incubated for 5 minutes in transfer buffer. Membrane was then placed onto the whatman paper, gel was carefully layed on top. Air bubbles were removed, ensuring elictricity flow. After coverage by 3 further layers of whatman, blotting was performed at 200 mA for 1 hour.

Blocking:
In order to minimize nonspecific antibody interactions, membrane was incubated in blocking buffer (TBS (pH 7.5), 5% milk powder) for one hour, shaking.

Primary antibody incubation:
A 1:10 dilution of the B1 antibody in TBS + 1% milk was prepared. Membrane was incubated in 50 mL falcon over night at 4°C, rotating.

Midi-Prep

Investigator: Chris W.

Midi-Prep of:


pSB1C3_CMV_VP123_453_Z34C =P832 =B656
pSB1C3_CMV_VP123_453_RGD_HSPG-KO =P833 =B658
pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 1 =P834 =B561
pSB1C3_001_RC_IRCK_P5tataless clone 1 =P835 =B516
pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P836 =B200
pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P837 =B200



The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no. P832P833P834P835P836P837
concentration (ng/µl)3615,8 1504,7 xxxx



Comment: P834-837 given to sven


Repetiton: Test digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI_hgH_rITR

Investigator: Stefan

Comment: Repetiton of yesterday's test digestion which yielded no clear band at 2000 bp as it was expected.

Components P822/P823 / µl
DNA 1,5
Buffer 4 1
BSA (10x) 1
SpeI 0,3
XbaI 0,3
H2O 5,9
Total volume 10


Gel:
0,45g agarose, 50 ml TAE (0,9%), 3 µl GELRED, 90 Volt, running time ~110 minutes

Freiburg10 td131010.jpg

Comment: This time both samples look well. P822 will be sent for sequencing.

Sequencing analysis: pSB1C3_leftITR_pCMV_betaglobin_mGMK_TK30_hgH_rITR (P825)

Investigator: Stefan

For sequencing the following primers were used_

  • qPCR primer CMV for (O20)
  • GOI primer for (O30)
  • seq. primer TK GMK for (O23)
  • TK30 SDM Pst for (O191)

Sequencing comfirmed that the pysical sequence fits our theoretical. Next step will be to remove the remaining PstI restriction site within TK30.

Repetition of cloning of Rep into Rep78

Investigator: Kira
Comment: The last 2 approaches to digest Rep78 and to clone failed because Rep78 was almost gone after PCR purification and was not detectable on the gel, even if I used the double amount of DNA. Thus, new over night culture was grown in order to perform the experiment again

Regarding the activity conditions of the enzymes, digestion was performed in 2 steps.

Components Rep78
DNA 3 µl
BSA (100x) 2 µl
Buffer no. 3 2,0 µl
Enzyme SwaI 1,0 µl
H2O 12 µl
Total volume 20


incubation @ 25 C for approx. 2 h
Dephosphorylation was performed because of the blunt cleavage. PCR phosphorylation followed in order to get rid off the buffer because Buffer 3 is not appropriate for further digestion.

Components Rep78
DNA 30 µl
BSA (100x) 0 µl
Buffer no. 2 4,5 µl
Enzyme HindIII 1,0 µl
H2O 9,5 µl
Total volume 45


incubation for 2h at 37C

File:Rep78 2010 10 13.jpg

Ligation with T4 ligase

v(insert)=4,4 ul
v(vector)= 3,6 ul
T4 buffer= 1 ul
T4 ligase= 1 ul

incubation at RT for 45 min

Transformation according to the standard protocol with BL21

Midi-Prep

Investigator: Chris W.

Midi-Prep of:


pSB1C3_CMV_VP123_capins =P862 =B474
pSB1C3_P40_VP123 =P863 =B651



The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no. P862P863
concentration (ng/µl)3310,5 2209,2



149. labday 14.10.2010

Mini-Prep and test digestion of several constructs

Investigator: Stefan

Glycerol stocks were prepared:


Mini-Prep was performed according to standard protocol:

Test digestion:

Components P858 + P859 / µl P860 + P861 / µl
DNA 7 1,5
Buffer 4 1 1
BSA (10x) 1 1
Enzyme I PstI 0,3 BstXI 0,3
Enzyme II EcoRI 0,3 XbaI 0,3
Enzyme III - SpeI 0,3
H2O 0,4 5
Total volume 10 10

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

Freiburg10 td141010.jpg

Comment: .

Continuation of Western Blot of unmodified AAV2 capsids

Investigator: Hanna

Comment: Yesterday, SDS PAGE, Western Blot and primary B1 mouse antibody incubation was performed.
Today, secondary antibody incubation and ECL analysis will be done.


Because of these dissatisfying results SDS PAGE will be repeated. Instead of performing Western Blot, gel wioll be stained by Coomassie in order to optimize SDS-PAGE conditions.

Cloning hGH_rITR into (form P728) into pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI (P822)

Investigator Patrick

Digestions, 1 h 40 minutes, 37 °C:

Expected results for the 1% agarose gel:


Freiburg10 1014pat.jpg

As usual the PSB1C3 bands indicate a higher bp amount than they actually got.
The gelextraction ...


... and following ligation (2 µl Insert, 6 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer, 40 minutes, RT) were performed according to the standard protocol. After the transformation (with BL21) the cells were plated and put into the 37°C room.

Cloning of the ViralBricks 453-empty and 587 empty

These ViralBricks were not ordered as hybridized oligos, but can be cloned from the Cap-Gene-Synthesis into the ViralBrick MCS.

components volume of p320 (ViralBrick-BLA) /µl volume of p320 (ViralBrick-BLA) /µl volume of p668 (pSB1C3_RepCap_IRCK) /µl volume of p668 (pSB1C3_RepCap_IRCK) /µl
DNA 2µl 2µl 7µl 7µl
BSA (100x) 2.5µl 2.5µl 2.5µl 2.5µl
Buffer 4 (10x) 2.5µl 2.5µl 2.5µl 2.5µl
SspI 1µl 0 1µl 0
SalI 1µl 0 1µl 0
BamHI 0 1µl 0 1µl
PvuII 0 1µl 0 1µl
H2O 16µl 16µl 11µl 11µl
Total volume 25µl 25µl 25µl 25µl
  • Incubation: 4h


    Sample Marker Sample ViralBrick_BLA 453 cut Sample ViralBrick_BLA 587 cut Sample pSB1C3_001_RepCap_IRCK 453 cut Sample pSB1C3_001_RepCap_IRCK 587 cut
    Lane 7µl 30µl 30µl 30µl 30µl

    Freiburg10 ViralBrick 453 empty and 587 empty.jpg



    SDS PAGE of viral capsids

    Investigator: Hanna
    Comment: In order to optimize SDS PAGE with the viral capsid proteins, I didn't perform Western Blot, but Coomassie staining.

    1.: Preparation of 10% polyacrylamide gel:
    Following standard protocol.

    2.: Preparation of virus samples:
    Two different virus stocks with unmodified capsid were taken. 2 samples were prepared of each: One sample was untreated, the other one was centrifuged in order to sediment cell fragments.
    10 µL Laemmli buffer (5x) was added to 40 µL virus sample and incubated at 95°C for 5 minutes.

    3.: SDS PAGE
    Loading plan:

    10 µL PageRuler Prestained Protein Ladder10 µL Sample 1 (not centrifuged)20 µL Sample 1 (not centrifuged)10 µL Sample 1 (centrifuged)20 µL Sample 1 (centrifuged)10 µL Sample 2 (not centrifuged)20 µL Sample 2 (not centrifuged)10 µL Sample 2 (centrifuged)20 µL Sample 2 (centrifuged)


    Gel was run 1.5 hours at 10 mA.

    4.: Coomassie staining
    Gel was put into coomassie bath, heated in microwave and incubated for 1 hour, shaking.
    In order to remove background coomassie staining, gel was incubated in acetic acid (20%) for 2 hours, shaking.

    SDS PAGE of AAV2 stock - unmodified capsids


    Results revealed that samples ran well (vertically :) ) - 20 µL seems to be too much. Interestingly centrifuging the virus stocks in order to remove cell debris didn't lead to better results.

    To do: Perform Western Blot again - with 10 µL Virus stock, 0.5% instead of just 0.2% TBS-milk and longer washing steps.

    Sequence analysis of super constructs

    Investigator: Hanna
    Comment: We assembled targeting super constructs which specifically bind the EGF receptor with VP2 N-terminal fused ZEGFR:1907 Affibody and can either be purified via a loop inserted His-Tag or visualized via Streptavidin coupled fluorescent proteins.
    Further on these loop motifs were vloned to the "mVenus VP1 insertion".

    Freiburg10 Super BAP.png
    Freiburg10 Super His.png

    Freiburg10 VP1 BAP.png
    Freiburg10 VP1 His.png





























    Sequencing results showed that insertion of these constructs into pSB1C3_CMV worked!

    Next steps: Put sample sinto BioBrick Box.
    Midi-Prep + Transfection.

    Transfection with pCMV, p40, pAAV_RC

    Investigator: Kira
    Comment: Transfection was performed in order to evaluate the promoter activities.

    Yesterday, the cells were seeded in the 6 well plate by Sven. Transformation was performed according to the manufacturer protocol (Qiagen).

    Trafo evaluation of Rep78

    Investigator: Kira
    Transformation was successful. 2 clones will be picked for mini prep tomorrow.

    Picking clones

    Investigator: Kira

    Rep78 and Rep52 were picked (2 clones from each plate), in order to continue with mini prep and PCR amplification.

    Cloning of CD into pSB1C3_hGH_rITR

    Investigator: Stefan

    .


    Vector name:
    pSB1C3_hGH_rITR (P728)

    Insert name:
    pSB1C3_CD_SDM-PstI_III clone 1 (P858)
    pSB1C3_CD_SDM-PstI_III clone 2 (P859)

    Digestion:


    components volume P858 + P859 /µl volume P728 /µl
    DNA 6 8
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme EcoI11
    Enzyme XbaI-1
    Enzyme SpeI1-
    H2O86
    Total volume (e.g. 15,20,25,30 µl) 20 20


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



    Freiburg10 141010.jpg


    Gel extraction:
    Was performed according to protocol.


    T4 Ligation:

    ligation name 728 + 858728 + 859
    volume of vector 3,143,14
    volume of insert4,864,86
    T4 ligase buffer (10x)11
    T4 ligase 11


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

    150. labday 15.10.2010

    Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)

    Investigator Patrick
    Digestions, 2 h 10 minutes, 37 °C:

    • P666: 5 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl Xba, 1 µl PstI, 9 µl H2O
    • P729: 4 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl SpeI, 1 µl PstI, 10 µl H2O

    Expected results for the 1% agarose gel:

    • P666: about 2100 and 750 bp
    • P729: about 3300 and 20 bp


    Freiburg10 1015pat fertig.jpg

    The gelextraction ...

    P728: 11,8 ng/µl
    P822: 34,6 ng/µl

    ... and following ligation (2,5 µl Insert, 5,5 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer (10x), 40 minutes, RT) were performed according to the standard protocol. After the transformation (with XL1B) the cells were plated and put into the 37°C room. Two additional transformations were performed with ligations from Volker labeled: "ligation viral brick 453 empty" & "viral brick 587 empty".

    The following day the plates were checked for clones. Unfortunately there grew no clones on these two plates contrary to my plate with a a lot of clones.

    Midi-Prep

    Investigator: Chris W.

    Midi-Prep of:


    pSB1C3_001_RC_IRCK_P5tataless clone 1 =P866 =B516
    pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 =P867 =B526
    pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 =P868 =B529
    pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_BAP clone 1 =P869 =B680
    pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis clone 1 =P870 =B200



    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    plasmid-no. P866P867P868P869P870
    concentration (ng/µl)899,80 954,46 406,971642,761585,12



    mini prep of several constructs

    Investigator: Kira

    c(rep52_1)=299,04 ng/ul
    c(rep52_2)=290,07 ng/ul
    c(rep78_1)=142,32 ng/ul
    c(rep78_2)=175,36 ng/ul

    Cell culture

    Investigator: Kira

    The cells are still alive. Medium was exchanged.--> RNA will be harvested tomorrow


    Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR

    Investigator: Stefan

    Glycerol stocks were prepared:

    • B694 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1
    • B695 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2

    Mini-Prep was performed according to standard protocol:

    • P875 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1 c = 73,1 ng/µl
    • P876 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2 c = 78,3 ng/µl

    Test digestion:

    Components P875 + P876 / µl
    DNA 4
    Buffer 4 1
    BSA (10x) 1
    XbaI 0,3
    AgeI 0,3
    H2O 3,4
    Total volume 10

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

    Freiburg10 td151010.jpg

    Comment: Test digestion looks allright, cloning will be continued using P876.

    151. labday 16.10.2010

    Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR

    Investigator: Achim

    Plasmids:

    • P729: pSB1C3_lITR_CMV_ß-Globin
      • c= 243.4 ng/µl
    • P730: pSB1C3_lITR_pHTERT_ß-Globin
      • c= 81.1 ng/µl
    • P876: pSB1C3_CD_SDM-PstI_hGH_rITR
      • c= 78.3 ng/µl

    Digestion:

    components I1 (P729) I2 (P730) V (P876)
    DNA 6 1414
    BSA (10x) 2 22
    Buffer 4 (10x)2 2 2
    Enzyme EcoI11 1
    Enzyme XbaI-- 1
    Enzyme SpeI11 -
    H2O8- -
    Total 20 20 20

    Digestion: 2h, 37°C

    Prep. gel:

    • 0,8%, run for 45 min
    Expected Bands: I1: 1335, I2: 1138, V: 4000
    • Corresponding bands were cut out

    Gel ex.

    • Nanodrop concentrations:
      • I1: 37.54 ng/µl
      • I2: 25.38 ng/µl
      • V: 26.47 ng/µl

    Ligation:

    ligation name I1 + VI2 + V
    volume of vector 4.694.23
    volume of insert3.313.77
    T4 ligase buffer (10x)11
    T4 ligase 11
    • Ligation @ RT for 40 min

    Trafo:

    • Done by Kira



    Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI

    Investigator Patrick

    Yielded concentrations & given numbers:

    • pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1: 208,4 ng/µl , P877 / B696
    • pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2: 251,6 ng/µl , P878 / B696
    • pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 3: 212,6 ng/µl , P889 / B696



    Test-digestion: 0,5 µl SpeI, 0,5 µl PstI, 3 µl DNA, 1 µl Buffer 4, 1 µl BSA, 4 µl H2O, 40 minutes, 37°C
    Expected results: fragments with about 650 and 4900 bp
    Freiburg10 1016pat test.jpg
    Obviously, this test digestion has to be repeated.

    Preparations for tomorrow:

    • Mini-prep of 3 mutual pSB1C3_leftITR_CMV_beta-globin_CFP clones (have to be picked from the plate)
    • Midi-prep of pHelper
    • Midi-prep of B689:pSB1C3_lITR_CMV_betaglobin_mGMK_TK30_SDM-PstI_hGH_rITR clone 2

    Biobrick assembly of Rep78 and Rep52

    Investigator: Kira
    Comment: After replacing the mutated Rep parts by the ordered Rep parts, PCR amplification has to be done in order to produce a biobrick. PCR program:

    c(Rep52)=299 ng/ul
    c(Rep78)=175 ng/ul

    Rep52: praefix 094 & suffix 097
    Rep78: praefix 093 & suffix 097


    components volume in µl
    5x Phusion HF buffer 10
    10 mM dNTP mix 1
    primer_for (1:10 dilution) 2,5
    primer_rev (1:10 dilution) 2,5
    DNA template (1:100) 0,5
    DMSO 0,5
    Phusion polymerase 0,5
    H2O 32,5
    Total volume (e.g. 50 µl) 50


    CyclesTemperatureTime
    98°C30 sec
    10x98°C15 sec
    63°C25 sec
    72°C32 sec
    20x98°C15 sec
    66°C25 sec
    72°C32 sec
    1x72°C5 min
    Hold 4°C


    1% agarose gel
    Freiburg10 Rep78&Rep52 PCR.jpg

    Digestion of plasmid backbone:

    pSB1C3_001 is used as backbone

    Components <b>vector Volume/µL
    DNA 3,5 µl
    BSA (10x) 2 µl
    Buffer no. 4 (10x) 2,0 µl
    Enzyme 1 EcoRI-HF 0,5 µl
    Enzyme 2 SpeI 1,0 µl
    H2O 15 µl
    Total volume 25


    incubation @ 37 C for approx. 2 h

    1% agarose gel
    Freiburg10 digestion pSB1C31 16.10.jpg

    Digestion of PCR product:

    Components PCR product Volume/µL
    DNA 35,0 µl
    BSA (100x) 0,45 µl
    Buffer no. 4 4,5 µl
    Enzyme 1 EcoRI-HF 1,5 µl
    Enzyme 2 SpeI 2,0 µl
    H2O 1,5 µl
    Total volume 45


    incubation @ 37 C for approx. 2 h

    T4 ligation for 40 min
    Transformation according to the standard protocol

    RNA harvesting

    Investigator: Kira
    After transfection, the cells were incubated for 48 hours. Today, the cells will be harvested and RNA extracted, in order to perform RT-PCR and an additional PCR for evaluation of promoter activity.

    The transfected cells were trypsinised and centrifuged for 2 min. The supernatant was discarded and pellet washed 2x with PBS. RNeasy Kit [Qiagen] was used for RNA extraction according to the manufacturer protocol.

    c(CMV)= 335,69 ng/ul
    c(P40)= 857,92 ng/ul
    c(AAV_RC)= 760,21 ng/ul

    152. labday 17.10.2010

    Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP

    Investigator: Anna

    Vector name:
    pSB1C3_lITR_CMV_betaglobin_CFP_cl1 (P880): c = 452,67 ng/µl
    pSB1C3_lITR_CMV_betaglobin_CFP_cl2 (P881): c = 288,88 ng/µl
    pSB1C3_lITR_CMV_betaglobin_CFP_cl3 (P882): c = 288,36 ng/µl

    Test Digestion:

    components volume P880 - P882 /µl volume P434 /µl
    DNA 2 2
    BSA (10x) 1 1
    Buffer 4 (10x)1 1
    Enzyme NgoMIV0,30,3
    Enzyme AgeI0,30,3
    H2O5,45,4
    Total volume 10 10


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



    Freiburg10 Test digestion of pSB1C3 lITR CMV ß-globin CFP.jpg

    Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP

    Investigator: Stefan

    Cloning of our last GOI!


    Vector name:
    pSB1C3_lITR_CMV_betaglobin_CFP cl 1-3 (P880-P882)

    Insert name:
    pSB1C3_hGH_rITR (P728)

    Digestion:


    components volume P880 - P882 /µl volume P728 /µl
    DNA 3 8
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme PstI11
    Enzyme XbaI-1
    Enzyme SpeI1-
    H2O114
    Total volume (e.g. 15,20,25,30 µl) 20 20


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



    Freiburg10 171010.jpg

    Test digestion of all constructs looked alright, therefore, cloning was continued using P881 only.



    Gel extraction:
    Was performed according to protocol.


    T4 Ligation:

    ligation name 728 + 881
    volume of vector 2,68
    volume of insert5,32
    T4 ligase buffer (10x)1
    T4 ligase 1


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

    RT-PCR

    Investigator: Kira
    For further experiments, RNA has to be translated into cDNA. The PCR was performed according to the manufacturer protocol.

    153. labday 18.10.2010

    Mini-Prep and test digestion of several constructs

    Investigator: Jessica

    Glycerol stocks were prepared:

    • B702 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1
    • B703 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2
    • B704 = pSB1C3_001_VP3 clone 1
    • B705 = pSB1C3_001_VP3 clone 2
    • B706 = pSB1C3_001_VP2 clone 1
    • B707 = pSB1C3_001_VP2 clone 2
    • B708 = pSB1C3_001_Rep78 clone 1
    • B709 = pSB1C3_001_Rep78 clone 2
    • B710 = pSB1C3_001_Rep52 clone 1
    • B711 = pSB1C3_001_Rep52 clone 2
    • B712 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1
    • B713 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2
    • B714 = pSB1C3_001_VP1 clone 1
    • B715 = pSB1C3_001_VP1 clone 2

    Mini-Prep was performed according to standard protocol:

    • P886 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 c= 232,2ng/µl
    • P887 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2 c= 186,2ng/µl
    • P888 = pSB1C3_001_VP3 clone 1 c= 300,8ng/µl
    • P889 = pSB1C3_001_VP3 clone 2 c= 284,4ng/µl
    • P890 = pSB1C3_001_VP2 clone 1 c= 298,3ng/µl
    • P891 = pSB1C3_001_VP2 clone 2 c= 299,9ng/µl
    • P892 = pSB1C3_001_Rep78 clone 1 c= 143,9ng/µl
    • P893 = pSB1C3_001_Rep78 clone 2 c= 163,4ng/µl
    • P894 = pSB1C3_001_Rep52 clone 1 c= 166,6ng/µl
    • P895 = pSB1C3_001_Rep52 clone 2 c= 181,6ng/µl
    • P896 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 c= 250,5ng/µl
    • P897 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2 c= 173,4ng/µl
    • P898 = pSB1C3_001_VP1 clone 1 c= 272,7ng/µl
    • P899 = pSB1C3_001_VP1 clone 2 c= 294,8ng/µl
    • P900 = pSB1C3_hGH_rITR (from B160) c= 136,7ng/µl

    Test digestion:

    Components P886,887,892,893,894,895,896,897 / µl P888,889,890,891898,899 / µl
    DNA 1,5 1,5
    Buffer (4) 1 (2) 1
    BSA (10x) 1 1
    NgoMIV 0,4 -
    XbaI 0,4 -
    PstI - 0,6
    XcmI - 0,4
    H2O 4,5 4,5
    Total volume 10 10

    Gel:
    1,0g agarose, 100 ml TAE (1%), 6 µl GELRED, Volt, running time minutes

    Freiburg10 test digestion 886-899.jpg

    Comment:

    PCR of mGMK and SR39

    Investigator: Anna


    Vector name:
    pSB1C3_lITR_CMV_betaglobin_CFP cl 1-3 (P880-P882)

    Insert name:
    pSB1C3_hGH_rITR (P728)

    Digestion:


    components volume P880 - P882 /µl volume P728 /µl
    DNA 3 8
    BSA (10x) 2 2
    Buffer 4 (10x)2 2
    Enzyme PstI11
    Enzyme XbaI-1
    Enzyme SpeI1-
    H2O114
    Total volume (e.g. 15,20,25,30 µl) 20 20


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



    Freiburg10 171010.jpg

    Test digestion of all constructs looked alright, therefore, cloning was continued using P881 only.



    Gel extraction:
    Was performed according to protocol.


    T4 Ligation:

    ligation name 728 + 881
    volume of vector 2,68
    volume of insert5,32
    T4 ligase buffer (10x)1
    T4 ligase 1


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

    154. labday 19.10.2010

    155. labday 20.10.2010

    => Go to Labjournal October part 3 (labday 156 - 166 )

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