Team:Freiburg Bioware/NoteBook/Labjournal/September

• Plasmid: pCerulean_VP1up_NLS
• Plasmid number: P365
• Tube name: SB1
• Primer used: CMV-F

Mini-Prep and test digestion of pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

Investigator: Hanna

Comment: Zegfr:1907 (Affibody) and the His-Tag which was coupled to the middle linker were cloned into pCerulean. 3 clones of each construct were picked yesterday.

Plasmid Mini-Prep

• new vector name: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

Glycerol Stocks
1. pCerulean_Zegfr:1907

2. pCerulean_6xHis_MiddleLinker

Test digestion

• buffer used: 4; Restriction-enzymes used: Enzyme 1 PstI-HF ; Enzyme 2 EcoRI-HF
• Plasmid
  • Given Plasmid-Number: 371; DNA concentration: 381.3 ng/µL;
  • Given Plasmid-Number: 372; DNA concentration: 377.5 ng/µL;
  • Given Plasmid-Number: 373; DNA concentration: 409.7 ng/µL;
  • Given Plasmid-Number: 374; DNA concentration: 404.2 ng/µL;
  • Given Plasmid-Number: 375; DNA concentration: 366.1 ng/µL;
  • Given Plasmid-Number: 376; DNA concentration: 375.0 ng/µL;

Comments::)

Pipetting scheme for each test digestion:

• Incubation: 55 minutes

Agarose-Gel:

0.875 g Agarose, 50 mL TAE (1.75 %), 3 µL EthBr, at 115 Volt, running time: 35 minutes

• Marker: GeneRuler ladder mix

Comments: Test digestion looked well:


Clone 1 of each construct were sent for sequencing (P371 and P374 = HW1 and HW2). Used primer: GATC_std_CMV-F.

Midi-Prep of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1 & pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1

Investigators: Chris W.

Midi-Preps of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1=P377 and pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1=P378

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

Mini-Prep and test digestion of the CD biobricks

Investigator: Kira

Ohne Worte *lach*

Wh did you digest with eco and ndeI?? is there a spechía reasonn for it?? if the coonstruct was in the rfc standard could digest it with the igem standard enzymes??

109. labday 03.09.2010

Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR

Investigator: Achim

Comment: The test digestion did not show the expected fragments of 2000 and 2500 bp. the photo was exposed too long to see distinct bands. Digestion will be repeated.

Sequencing results

Investigator: Hanna

Comment: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker were cloned and sent for sequencing yesterday.

1. pCerulean_Zegfr:1907: Sequencing looked well.

2. pCerulean_6xHis_MiddleLinker: Sequencing looked also well.

Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Investigator: Hanna

Comment: For N-terminal fusion to VP2, the Affibody - fused to different kinds of linkers - will be cloned into the expression plasmid pCerulean. For imaging CFP, which was fused to the middle linker, will be cloned into pCerulean.

Practical Cloning:

• plasmid:
  • Vector: name: pCerulean number: P273
  • Insert: name: pSB1C3_Zegfr:1907_LongLinker (P298), pSB1C3_Zegfr:1907_MiddleLinker (P290), pSB1C3_Zegfr:1907_SEG (P296), pSB1C3_Zegfr:1907_ShortLinker (P292), pSB1C3_CFP_MiddleLinker (P276)
• new vector name: pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker
• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 EcoRI-HF ; Enzyme 2 PstI-HF
• DNA concentration (vector): 419.5 ng/µL ; DNA concentration (insert): P298 229.7 ng/µL, P290 227.4 ng/µl; P296 218.7 ng/µL; P292 196.6 ng/µL; P276 207.7 ng/µL

Comments: No.

Digestion

• Incubation: 1.5 h

Agarose-Gel:

0.8 g Agarose, 50 TAE (1.5 %), 5 µL EthBr, at 70 Volt, running time: ~ 1 hour

• Marker: GeneRuler ladder mix

Gel extraction

Gel measurement:

Ligation

1. PCerulean_Zegfr:1907_LongLinker

2. PCerulean_Zegfr:1907_MiddleLinker

3. PCerulean_Zegfr:1907_SEG

4. PCerulean_Zegfr:1907_ShortLinker

5. PCerulean_CFP_MiddleLinker

Trafo

Trafo was performed following the standard protocol. Used cells: XL1b, DNA amount: 2.5 µL of each ligation reaction. Plates were stored @ 37°C room over night.

Harvest viral particles, Seeding HT1080 cells

Investigator: Adrian, Kerstin

• Harvest viral particles of Transfection from 31.08.2010 (six approaches: 10µg of RC (2x P326 and 2x P325 and 2x Stratagene), pHelper and GOI (P262))
• Seeding HT1080 cells

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Investigator: Patrick

The Miniprep was performed according to the standard protocol. Labelling:

• P381: pCerulean_middlelinker clone 1 upper band : 526,0 ng/µl
• P382: pCerulean_middlelinker clone 2 upper band : 522,0 ng/µl
• P383: pCerulean_middlelinker clone 3 upper band : 463,0 ng/µl
• P384: pCerulean_middlelinker clone 1 lower band : 465,0 ng/µl
• P385: pCerulean_middlelinker clone 2 lower band : 500,0 ng/µl
• P386: pCerulean_middlelinker clone 3 lower band : 524,0 ng/µl

Öhm Patrick: Hier ist der Grund, warum das Experiment "wiederholt" wurde: Falsche Beschrieftung hier, als auch in der Excel Tabelle ;) "Finde den Fehler!" :P

see also http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Cloning_CFP_middlelinker_.28from_pSB1C3_CFP_middlelinker.2C_P276.29_into_pCerulean_.28P273.29

;-) bitte ausfüllen

Testdigestion: 7 µl DNA sample, 1 µl Buffer 4 (10x), 1 µl BSA, 0,5 µl Xba, 0,5 µl PstI-HF
Expected size of the fragments: 786 & 2053bp
There seems to be something wrong.

Sent for sequencing: P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev
Used primer: O21 : CMV_reverse_qPCR

Cloning of pSB1C3_leftITR_pTert and pSB1C3_mGMK with pSB1C3_leftITR_CMV

Investigator: Chris L.

• Vector: name: pSB1C3_leftITR_pTERT clone 1 P256 c=179,2 ng/µl
• Vector: name: pSB1C3_leftITR_CMV P188 c=231,6 ng/µl
• Insert: name: pSB1C3_mGMK P195 c=258,6 ng/µl
• new vector name: pSB1C3_leftITR_CMV_GMK
• new vector name: pSB1C3_leftITR_pTERT_GMK
• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 PstI ; Enzyme 2 SpeI ; Enzyme 3 XbaI

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45

Results:

• c(Insert)= 15,37 ng/µl; size: 627 bp
  
• c(Vector)= 50,84 ng/µl; size: 2869 bp
• c(Vector)= 75,64 ng/µl; size: 2672 bp

• Ligation of PCR products and vector:

For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used. Incubation time: 45 min

• Transformation:

The transformation was done following the standard protocol using XL1 blue cells.

Sequence analysis of pCerulean_VP1up

Investigator: Bea

The assembled pCerulean_VP1up which serves as the first construct in the VP1 insertion cloning assembly was sent for sequencing.

• Plasmid used: P365
• Primer used: CMV-F
• Tube name: SB1
• Folder (Geneious): N-terminal Targeting --> pCerulean_VP1up_NLS

pCerulean_VP1up_NLS_Targeting molecule

Investigator: Bea

Comment: For preparing the next step in the VP1 insertion, different targeting molecules will be fused to the construct pCerulean_VP1up_NLS.

1. The first construct is the Affibody Z_EGF-R:1907 which was already cloned into the standard iGEM plasmid and can be sued for target tumor cells overexpressing the EGF receptor.
2. The second construct is the mVenus (Yellow fluorescent protein) which can be used for imaging experiments
3. Another motif will be the His-tag. Since the His-tag is very small, the oligos will be hybridized and directly ligated into the pCerulean backbone.

Digestion of vector:

• Plasmid used: pCerulean_VP1up_NLS (P365) c=470 ng/µL
• Add BSA
• AgeI-HF and SpeI-HF were used

Protocol of the digestion of the vector:

• Incubation of the sample at 37°C
• Incubation for 120 minutes

The Targeting molecules were digested with different enzmyes than the vector for providing the ability to assemble the different constructs together without creating new restriction site but to delete them. This works by fusing NgoMIv and AgeI together.

Digestion of the targeting molecules

• Plasmids used:
1. pSB1C3_zEGF-R:1907 (P284) c=146 ng/µL
2. pGA14_mVenus(P60) c=280 ng/µL
• Add BSA
• NgoMIV and SpeI-HF were used

• Incubation of the sample at 37°C
• Incubation for 90 minutes

After incubation the samples were loaded on a 1% agarose gel. The obtained gel (after running the gel for 15 minutes) can be seen below.

Results: The boxes mark fragments which ere cut out of the gel. The left lane (pCerulean) looks a bit strange, because Marker was added after the digestion reaction instead of Loading dye.

Parallel, for assembling the His-Tag to the construct pCerulean_VP1up_NLS, oligos for the His-Tag in the RFC25 standard (provided by Gerrit) were hybridized and can directly be ligated in the digested vector. This strategy was used because of the smal size of the His-Tag which cannot be separated easily by the agarose gel.

Hybridization of the His-Oligos

• 10µL oligo1 (1:10)
• 10µL oligo2 (1:10)
• 4µL 100mM Tris-Cl pH 8
• 10µL 5mM MgCl₂
• 8µL H₂O

The used programm for the hybridization:

• 99°C 7´
• 99°C 1´
• -1°C R=0,3°/s
• Goto 2 rep 74
• Hold 4°C

After the gel extraction of a T4 ligation was performed.

Affibody Z_EGF-R:1907:

v_{Cerulean_VP1up_NLS} = 5,82µL
v_ZEGF-R:1907 = 2,18µL



mVenus Z:

v_{Cerulean_VP1up_NLS} = 4,04µL
v_mVenus = 3,96 µL



6xHisTag:

v_{Cerulean_VP1up_NLS} = 0,2µL
v_6xHisTag = 7,98µL

Next steps: Picking clones of the trafo plates (containing Kanamycin) and perform the Mini-Prep. The obtaining constructs finally will be fused to the VP2/3 protein which results in the final construct for the VP1 targeting approach.

3rd Repetition of Mini-Preps and test digestion for Loop insertion BioBricks

Investigator: Achim, Anna

Sequencing results of Loop insertion BioBricks from 02.09.10; the following clones looked well:

• pSB1C3_587_KO_RGD_clone6.4
  
• PSB1C3_587_KO_His_clone9.4

Update: 9 of the 14 ViralBricks looked well (sequencing results from 01. - 03.09.). Two new clones of the remaining constructs were prepared and test digested.

Test digestion:

Incubation time: 1 h, Incubation temperature: 37°

Preparation of gel:
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

Expected fragment sizes /bp:
pSB1C3: 2051 bp
BAP: ~150
Z34C: ~ 200

Comment: The following clones were sent for sequencing: 1.6, 12.6 ,13.6 and 14.5.
To do: Retrafo of clone6.4 and clone9.4 and preparation of glycerol stocks.

Cloning of Cap into pAAV_RC_ins-rep: preparation for SDM

Investigator: Stefan

Comment: Cloning Cap into pAAV still does not work as planned. As another approach Cap can be cut using BsiWI and XcmI and performing a SDM in the part of Cap not cloned into pAAV. Primers are ordered and should arrive on monday to proceed.

Digestion:

Comment:Digestion was performed using two steps, first incubating for 1 hour at 37 °C, afterwards for 1,5 hours at 55 °C.

Gel:

0,5 g Agarose,50 ml TAE (1%), 5 µl EtBr , at 115 Volt, running time: 50 minutes

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

• pMA_RepCap Vector_SDM_InsPvuII (P211): c= 6,26 ng/µl
• pAAV_RC_ins-rep (P250): c= 13,72 ng/µl

Quick Ligation:

Comment: XcmI produces only 1 base overhang, therefore Quickligase was used for ligation and incubation time increased.

The Ligation was performed as following:

• Vector Volume: 4,59 µl
• Insert Volume: 4,41 µl

• 10 µl QuickLigase buffer (2x)
• 9 µl (Vector + Insert) mix
• 1 µl QuickLigase

Incubating for 60 minutes.

Transformation:

Trafo was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

110. labday 04.09.2010

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273): Sequencing results of P381 and P385

Investigator:Patrick

Sequencing results of P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev: A wrong primer was used so there are no relevant sequence data yet. On sunday i will sent these clones again for sequencing. Primer: GATC_std_CMV-F

Ha ich war schneller als Bea beim "Kopfkratzen-setzen" *lol*

Hiermit verspreche ich dass es noch jede Menge Kopfkratzer, Laboraufsichtswichtel und pinke Panter geben wird (Patrick)

Harvest viral particles, Transduction

Investigator:Kerstin, Adrian

• Harvest viral paricles from one plate of transfection from 01.09.2010 (10 plates, same treatment (10µg of each plasmid P357, P263, P356))
• Transduction:

1. 1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P326)
2. 1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P325)
3. 4x 6-well: 10x 0,5ml and 4x 1ml of virus from 01.09.2010 (stratagene R/C)
4. 4x 6-well: 10x 0,5ml and 4x 1ml of virus from 04.09.2010 (10 plates same treatment)
5. 6x 6-well: 0,5ml of virus from 21.08.2010 (TKGMK; 18x pH 7,10 and 6x pH 7,12)

Repetition of Hybridisation and Cloning for Loop insertion BioBricks

Investigator: Achim, Anna

• Samples:

1: 453_BAP
11: 453_Z34C
12: 587_Z34C
13: 587_KO_Z34C
14: 587_KO_Z34C_Spacer

Comment: New approaches of the Hybridisation and Fill-in reactions were done because it didn't work the first time. A possible reason is that the klenow enzyme wasn't inactivated. In addition, the conditions for ligation will be improved. Also the ligation of 453_BAP was done again.

Digestion:

° 1 µl each

Preparation of gel:
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 50 minutes

Repetition: Prepatation for SDM: cloning of Cap into pAAV

Investigator: Stefan

Schönen Abend dir noch

Comment: On yesterday's plates nothing grew. Because gelex and ligation products were stored in coldroom, new ligation and trafo approaches were performed.

Ligation approaches:

• ligation with Quick Ligase (as performed yesterday)
• ligation with T4 Ligase

Transformation approaches:

• Quick Ligase (approach from yesterday):
  • 2 µl of ligation product
  • 4 µl of ligation product
    

• Quick Ligase (new approach):
  • 2 µl of ligation product
  • 4 µl of ligation product
    

• T4 Ligase:
  • 2 µl of ligation product
  • 4 µl of ligation product
    

Transformation:

Trafo of each approach was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

Ligation and trafo of the modified Cap insert , the Viral Bricks 1, 11, 12, 13 and 14

Investigator: Volker

Ligation

Preparation of PBS Buffer

Investigator: Volker

Two liters of PBS buffer were prepared for the column purification of the viral particles. The following protocol was used:

• dissolve the following in 800ml ddH₂O:
  • 8g of NaCl
  • 0.2g of KCl
  • 1.44g of Na₂HP0_$ (1.69 because of the crystal water in the reagent)
  • 0.24 g of KH₂PO₄
• adjust to pH 7.4
• adjust to 1000ml
• steril filtrate the solution

111. labday 05.09.2010

Mini-prep of glycerol stock pMA_RepCap Vector_SDM_InsPvuII clone 1 (B182)

Investigator: Stefan

Glycerol stock: Because glycerol stock B182 was not mixed before putting into -80 °C freezer the cells died. Therefore a new glycerol stock was prepared and labled B302.

Mini-Prep was performed according to the standard protocol. Two preps were prepared:

Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Investigator: Hanna

2 clones of each construct (1. pCerulean_Zegfr:1907_ShortLinker, 2. pCerulean_Zegfr:1907_MiddleLinker, 3. pCerulean_Zegfr:1907_LongLinker, 4. pCerulean_Zegfr:1907_SEG and 5. pCerulean_CFP_MiddleLinker) were picked.

To do tomorrow (6.9.): Mini-Preps and Test digestion (with EcoRI and PstI).

112. labday 06.09.2010

Minipreps & Test PCR of pTert Biobrick assembly

Investigator: Achim

• After unsucessful test digestion with EcoRI and PstI I tried out a test PCR with Igem Primers (same Mastermix as for loop inserts)
• Elongation time was set to 2.35 min because the amplified fragment should be about 2.5 kbp big.
• Result:

The visible bands are not specific. Either cloning or PCR went wrong :)

• Next steps: Another test digestion tomorrow, if that doesn't work out I'll repeat the ligation.

Minipreps & Test PCR of Loop inserts 1, 11, 12 ,13, 14

Investigator: Achim & Anna

• Samples:

1: 453_BAP
11: 453_Z34C
12: 587_Z34C
13: 587_KO_Z34C
14: 587_KO_Z34C_Spacer

• Minipreps of 1.1, 1.3, 11.1, 11.2, 12.1, 12.2, 13.1, 13.2, 14.1, 14.2
• Clones 1.4, 11.3, 11.4, 12.3, 12.4, 13.3, 13.4, 14.3, 14.4 were pelleted and stored away in case the first two are incorrect.

• In the hope to confirm our small inserts, A test PCR using Igem standard primers was carried out:

PCR-reaction:

PCR program:

Results:

Bands should be about 150 for BAP and about 200 for Z34C. Bands dont quite match the expectations, somehow the BAP band seems bigger than the Z34C band. Clones 1.3, 11.1, 12.2, 14.1 were sent for sequencing.

Mini-Preps and test digestion of Linker BioBricks

Investigator: Anna

Comment: Mini-preps of all clones were prepared. The have not been added to the excel sheets yet, but were stored in the freezer.

Test digestion:

Preparation of gel:

• Loading plan:

SL: Shortlinker ML: Middlelinker LL: Longlinker

• Incubation time: 1 h, Incubation temperature: 37°
  
• 1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

• Expected fragment sizes:
  

VP1up_NLS_Histag: 550 bp
ZeGFP:1907_SEG: 344 bp
ZeGFP:1907_ML: 261 bp
CFP_ML: ~ 1000 bp
ZeGFP:1907_SL: 249 bp
ZeGFP:1907_LL: 237 bp

To do: Test digestion of VP1up_NLS_mVenus and Affibody. Number 2, 3, 5, 7, 9 and 12 will be sent for sequencing tomorrow.

Production of Ampicilin and Chloramphenicol

Investigator: Jessica

• 10ml ethanol (70%) containing 1g Amp in 60µl aliquots
• 10ml ethanol (70%) containing 0,25g Cm in 60 µl aliquots

Mini-prep and test digestion of pAAV_RC-CapIns_prepSDM

Investigator: Stefan

Mini-Prep was performed according to the standard protocol

Sent for sequencing: Clone 3 (P396) was sent for sequencing.

• Tube name: SB1
• Primer used: VP 4200 rev

Sent for sequencing

Investigator: Stefan

• Tube name: SB2

pAAV_RC_inserts clone1 (P326) was sent for sequencing using the following primers:

• VP1 primer for pKex rev
• SK
• Primer Cap 2800 for
• Primer Cap 2800 rev
• Primer Cap 3500 for