Team:TUDelft/3 August 2009

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='''3 August 2009'''=
='''3 August 2009'''=
 +
 +
===Tim Weenink===
 +
 +
I stole p(tetR) promotor and double terminator from the delay team to redo the !B and !C assemblies. The ligation products were run on Calin's gel (see below). The transformants were plated on Cam agar plates (unfortunatele only LB agar was left that seemed infected.) and stored in the 37 degrees C stove.
 +
 +
===Sriram===
 +
All the transformations done on friday gave growth. However comparing to the chemically competent cells prepared by TMF buffer (RbCl) the number of colonies from TSS buffer competent cells was less. However the concentration of DNA added plays a major role in the efficiency of transformation. The λp-R-GFP-T DNA isolated by miniprep had a concentration of 116.5 ng/µl which gave huge number of colonies compared to the diluted pSB1K3 with mRFP biobrick. The electro-transformation of λp-rep biobrick was successful and gave good colonies. hence we have decided to work with electro-transformation hereafter.
 +
 +
Thus we can very well start the assemblies today. We did restriction for the DNA samples needed for 8 assemblies. We might have some problem with backbones but only in future assemblies.
 +
 +
[[image:Gel03082009-delay.png‎|center|thumb|545px]]
 +
 +
{| border="1" align="center"
 +
| Well No. || Sample Name || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1|| pTet-Upstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 2|| RBS-cI-RBS-Downstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3|| pSB1C3-plasmid || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4|| RFP-Upstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5|| Term-Downstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 6|| pSB1C3-plasmid || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 7|| pLacI-Upstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 8|| RBS-Downstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 9|| pSB1C3 plasmid || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 10|| [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 11|| TetR-Upstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 12|| Term-Downstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 13|| pSB1C3 plasmid || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 14|| pTet-Upstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 15|| Lock3C-Downstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 16|| pSB1C3 plasmid || || <font color=limegreen>&#10004;</font>
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|- align="center"
 +
| 17|| cI-Upstream || || <font color=limegreen>&#10004;</font>
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|- align="center"
 +
| 18|| Term-Downstream || || <font color=limegreen>&#10004;</font>
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|- align="center"
 +
| 19|| pSB1C3 plasmid || || <font color=limegreen>&#10004;</font>
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|- align="center"
 +
| 20|| pLambdain-Upstream || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 21|| RBS-GFT-T-Downstream || || <font color=limegreen>&#10004;</font>
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|- align="center"
 +
| 22|| pSB1C3 plasmid || || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 23|| [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>&#10004;</font>
 +
|}
===Calin===
===Calin===
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</html>
</html>
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Growth on all plates. The pSB4C5 plate was parafilmed and placed in fridge.
+
Growth on all plates. The pSB4C5 and R751 plates were parafilmed and placed in fridge.
 +
 
 +
 
 +
Did linearization of oriT-R, promoter, pSB1AK3, and pSB4C5 using EcoRI.
 +
 
 +
Did digests of oriT-R (9uL) and pSB4C5 (4.5uL).
 +
 
 +
Did ligation CA and CB:
 +
 
 +
{| border="1" align="center"
 +
| Assembly name || position || Components || volume in µl
 +
|- align="center"
 +
| CA|| Upstream || pTet-GFP || 2
 +
|- align="center"
 +
| || Downstream || oriT-R || 2
 +
|- align="center"
 +
| || Backbone || pSB4C5 || 2
 +
|- align="center"
 +
| ||  || H20 || 11
 +
 
 +
|- align="center"
 +
| CB|| Upstream || rbs-GFP-term ||2
 +
|- align="center"
 +
| || Downstream || oriT-R || 2
 +
|- align="center"
 +
| || Backbone || pSB1C3 || 2
 +
|- align="center"
 +
| ||  || H20 || 11
 +
|}
 +
 
 +
 
 +
Ran a 2% gel to check various parts and digests.
 +
 
 +
[[Image:Gel03082009-conj.png|center|thumb|545px]]
 +
 
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || oriT-R linear || 2079 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 2 || oriT-R old digest || 2079 + ~278  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3 || oriT-R control || 2079 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || oriT-R new digest || 2079 + ~278  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || promo linear || 2983 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 6 || promo control || 2983 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 7 ||  [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 8 || pSB1AK3 linear || 3864 ||  <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 9 || pSB1AK3 control || 3864 ||  <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 10 || pSB4C5 linear || 3896 ||  <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 11 || pSB4C5 control || 3896  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 12 || pTet digest || 2079 + 937 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 13 || GFP-gen digest || 2079 + 878 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 14 || pSB1C3 digest || 2072 + 675 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 15 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 16 || !B assembly ligation || circular || ?
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|- align="center"
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| 17 || !C assembly ligation || circular || ?
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|}
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===Orr===
 +
Made some LB Agar medium and some agarose gel with pre-made TBE at concentration 1x.
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Latest revision as of 07:19, 20 October 2009

Lab Notebook

July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
 

3 August 2009

Tim Weenink

I stole p(tetR) promotor and double terminator from the delay team to redo the !B and !C assemblies. The ligation products were run on Calin's gel (see below). The transformants were plated on Cam agar plates (unfortunatele only LB agar was left that seemed infected.) and stored in the 37 degrees C stove.

Sriram

All the transformations done on friday gave growth. However comparing to the chemically competent cells prepared by TMF buffer (RbCl) the number of colonies from TSS buffer competent cells was less. However the concentration of DNA added plays a major role in the efficiency of transformation. The λp-R-GFP-T DNA isolated by miniprep had a concentration of 116.5 ng/µl which gave huge number of colonies compared to the diluted pSB1K3 with mRFP biobrick. The electro-transformation of λp-rep biobrick was successful and gave good colonies. hence we have decided to work with electro-transformation hereafter.

Thus we can very well start the assemblies today. We did restriction for the DNA samples needed for 8 assemblies. We might have some problem with backbones but only in future assemblies.

Gel03082009-delay.png
Well No. Sample Name Expected Plasmid Size Status
1 pTet-Upstream ✔
2 RBS-cI-RBS-Downstream ✔
3 pSB1C3-plasmid ✔
4 RFP-Upstream ✔
5 Term-Downstream ✔
6 pSB1C3-plasmid ✔
7 pLacI-Upstream ✔
8 RBS-Downstream ✔
9 pSB1C3 plasmid ✔
10 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
11 TetR-Upstream ✔
12 Term-Downstream ✔
13 pSB1C3 plasmid ✔
14 pTet-Upstream ✔
15 Lock3C-Downstream ✔
16 pSB1C3 plasmid ✔
17 cI-Upstream ✔
18 Term-Downstream ✔
19 pSB1C3 plasmid ✔
20 pLambdain-Upstream ✔
21 RBS-GFT-T-Downstream ✔
22 pSB1C3 plasmid ✔
23 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔

Calin

Tim pointed out that the plasmids should be linearized before doing the gel. Here are possible digestions on some of the parts:

Backbone Part Unique Sites Present
pSB1A2 oriT-R AatII AccI AflIII BglI BspLU11I BstAPI
DrdI Eam1105I EcoRI FspI NspI PstI
PvuI RsaI ScaI SgrAI SpeI SspI
StyI TatI VspI XbaI XmnI ZraI
BBa_J61002 strong promoter AatII AlwNI AvrII BglI BsiWI BspLU11I
BstXI DrdI EcoRI FspI HaeII HpaI
NcoI NspI PflMI PshAI PstI PvuI
PvuII RsrII ScaI SgrAI SpeI SspI
TatI VspI XbaI XmnI ZraI
pSB1AK3 ccdB AatII AccB1I AccI BamHI BanII BglI
BspLU11I BsrGI BssHII BstXI BstZ17I BtrI
ClaI DraIII DrdI Eam1105I EcoNI EcoRI
FspI HaeII HincII HindIII MscI
NruI NspI PflMI PstI ScaI SgfI
SpeI SrfI XbaI XmnI ZraI
pSB4C5 ccdB AatII AccI AclI AflII ApaLI BamHI
BbeI BseRI BsrGI BssHII BstAPI BstXI
BstZ17I BtrI Cfr10I EcoRI EcoRV HaeII
HincII KasI NarI NdeI PstI
PvuII SacI ScaI SfoI SmaI SpeI
SphI SrfI StyI XbaI XmaI ZraI

Growth on all plates. The pSB4C5 and R751 plates were parafilmed and placed in fridge.


Did linearization of oriT-R, promoter, pSB1AK3, and pSB4C5 using EcoRI.

Did digests of oriT-R (9uL) and pSB4C5 (4.5uL).

Did ligation CA and CB:

Assembly name position Components volume in µl
CA Upstream pTet-GFP 2
Downstream oriT-R 2
Backbone pSB4C5 2
H20 11
CB Upstream rbs-GFP-term 2
Downstream oriT-R 2
Backbone pSB1C3 2
H20 11


Ran a 2% gel to check various parts and digests.

Gel03082009-conj.png
Well Part Expected Plasmid Size Status
1 oriT-R linear 2079 ✔
2 oriT-R old digest 2079 + ~278 ✔
3 oriT-R control 2079 ✔
4 oriT-R new digest 2079 + ~278 ✔
5 promo linear 2983 ✔
6 promo control 2983 ✔
7 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
8 pSB1AK3 linear 3864 ✔
9 pSB1AK3 control 3864 ✔
10 pSB4C5 linear 3896 ✔
11 pSB4C5 control 3896 ✔
12 pTet digest 2079 + 937 ✔
13 GFP-gen digest 2079 + 878 ✔
14 pSB1C3 digest 2072 + 675 ✔
15 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
16  !B assembly ligation circular  ?
17  !C assembly ligation circular  ?

Orr

Made some LB Agar medium and some agarose gel with pre-made TBE at concentration 1x.

Retrieved from "http://2009.igem.org/Team:TUDelft/3_August_2009"