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From 2010.igem.org

Contents 1 136. labday 01.10.2010 1.1 mini preps of CD clones 1.2 test digestion of CD clones 1.3 Biobrick assembly pSB1C3_lITR_hTERT_beta-globin_CD 1.4 Sequencing results of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO 1.5 Picking clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO 1.6 Sequencing results of pSB1C3_001_VP2/3_587-KO_BAP and pSB1C3_001_VP2/3_587-KO_6xHis 1.7 Impressions of transfection of AAV293 with pCerulean_VP1up_NLS_mVenus_VP2/3 1.8 Cloning of VP2/3_BAP_HSPG-KO and VP2/3_His_HSPG-KO into pCerulean_Zegfr:1907_Middlelinker and VP2/3_His_HSPG-KO into pCerulean_VP1up_NLS_mVenus

136. labday 01.10.2010

mini preps of CD clones

Investigator: Kira

c(p689)= 115 ng/ul
c(690)= 151, 20 ng/ul
c(691)= 122,27 ng/ul
c(p692) = 126,42 ng/ul

test digestion of CD clones

Investigator: Kira

Components	sample Volume/µL
DNA	4,0 µl
BSA (10x)	2 µl
Buffer no. 4	2,0 µl
Enzyme 1 XbaI	1,0 µl
Enzyme 2 AgeI	1,5 µl
H2O	9,5 µl
Total volume	20

incubation @ 37 C for approx. 2 h

1% agarose gel

Biobrick assembly pSB1C3_lITR_hTERT_beta-globin_CD

Investigator: Kira

c(pSB1C3_lITR_hTERT_beta-globin)= 333 ng/ul
c(pSB1C3_CD)= 151 ng/ul

Components	vector Volume/µL	insert Volume/µL
DNA	4,5 µl	6
BSA (10x)	3 µl	3
Buffer no. 4	3,0 µl	3
Enzyme 1 XbaI	0 µl	1,5
Enzyme 2 SpeI	1,5 µl	0
Enzyme 3 PstI-HF	1,0	1
H2O	17	15,5
Total volume	25

incubation @ 37 C for approx. 2 h

1% agarose gel

Ligation

DNA-mix: 8 ul (vector 4,6ul)+(insert 3,4 ul)
T4 ligase: 1 ul
T4 buffer: 1 ul
Incubation @ RT for 30 min

Transformation was performed according to the standard protocol w BL21 cells.

Sequencing results of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna

Comment: All N-terminal fusion approaches with VP2/3_HSPG-KO revealed positive results except of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO. Another clone was picked, preped, test digested and sent for sequencing.

Conclusion: Sequencing results revealed positive results.

Picking clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna

Unfortunately there grew a bacteria lawn over night - it was hardly not possible to pick clones. Nevertheless I tried and picked 2 clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO.
To do: Mini-Prep and test digestion.

Sequencing results of pSB1C3_001_VP2/3_587-KO_BAP and pSB1C3_001_VP2/3_587-KO_6xHis

Investigator: Hanna

Comment: For the creation of our super constructs, the His-Tag and the BAP motif need to be cloned into VP2/3 for N-terminal fusion to VP2. Sequencing results showed that the 6xHis and BAP motif was not cloned into VP2/3_insCap.

Sample gallery

To do: Clone 587-KO_6xHis and 587-KO_BAP into pSB1C3_001_VP2/3_insCap 1. via digestion of inserts and 2. via hybridization of referring oligos - digestion of vector with 800-900 ng.

Impressions of transfection of AAV293 with pCerulean_VP1up_NLS_mVenus_VP2/3

Investigator: Adrian

Yesterday AAV293 cells were transfected with pCerulean_VP1up_NLS_mVenus_VP2/3 and GMK-TK30 as gene of interest. Today, nuclear localization of the produced mVenus_VP2/3 fusion protein was visible and can be nicely seen in the following pictures:

Conclusion: The VP2/3 fusion particle was transcribed and translated AND was transported back into the nucleus in order to be packaged by the ITR-flanked gene of interest.

Cloning of VP2/3_BAP_HSPG-KO and VP2/3_His_HSPG-KO into pCerulean_Zegfr:1907_Middlelinker and VP2/3_His_HSPG-KO into pCerulean_VP1up_NLS_mVenus

Investigator: Stefan

Vector name:

• pCerulean_Zegfr:1907_Middlelinker (P408)
• pCerulean_VP1up_NLS_mVenus (P426)

Insert name:

• pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone1 (P660)
• pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone2 (P661)
• pBS1C3_001_VP2/3_587_His_HSPG-KO clone1 (P662)
• pBS1C3_001_VP2/3_587_His_HSPG-KO clone2 (P663)

components	volume for inserts (P660 - P663) /µl	volume of P408 /µl	volume of P426 /µl
DNA	10	3	3
BSA (10x)	3	2	2
Buffer 4 (10x)	3	2	2
Enzyme NgoMIV	1	-	-
Enzyme PstI	1	1	1
Enzyme MscI	1	-	-
Enzyme AgeI	-	1	1
H2O	11	11	11
Total volume (e.g. 15,20,25,30 µl)	30	20	20

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

Gel extraction:
Was performed according to protocol.

T4 Ligation:

ligation name	408 + 660	408 + 661	408 + 662	408 + 663	426 + 662	426 + 663
volume of vector	3,4	3,05	3,35	2,56	2,74	3,13
volume of insert	4,6	4,95	4,65	5,44	5,26	4,87

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