Team:University of Lethbridge/Notebook/GeneralLabOctober

From 2008.igem.org

(Difference between revisions)
m (October 11, 2008)
m (spacing)
 
(8 intermediate revisions not shown)
Line 8: Line 8:
-the iGEM pLacI had a faint band, RFP looks good (half of it cut), TetR looks like it either didn't cut at all, or only cut once. Only used 2 uL for each sample, will try running with more.
-the iGEM pLacI had a faint band, RFP looks good (half of it cut), TetR looks like it either didn't cut at all, or only cut once. Only used 2 uL for each sample, will try running with more.
 +
===October 4, 2008===
===October 4, 2008===
Line 29: Line 30:
[[Image:Oct5gel.jpg | 250 px]]
[[Image:Oct5gel.jpg | 250 px]]
 +
===Ocotber 6, 2008===
===Ocotber 6, 2008===
Line 54: Line 56:
Plasmid prepped 3 mL of culture for various plasmids for the reporter system: pLacI, TetR sub, pLacI-3, RFP sub, and GFP sub. They are labelled with their name and today's date in 1.5 mL microfuge tubes (50 uL of elution solution). They are stored in the "iGEM Plasmids" box in the -20 C freezer.
Plasmid prepped 3 mL of culture for various plasmids for the reporter system: pLacI, TetR sub, pLacI-3, RFP sub, and GFP sub. They are labelled with their name and today's date in 1.5 mL microfuge tubes (50 uL of elution solution). They are stored in the "iGEM Plasmids" box in the -20 C freezer.
 +
===October 11, 2008===
===October 11, 2008===
====Roxanne====
====Roxanne====
-
-Ran a gel of the reporter system plasmids that Munima Isolated yesterday to determine to outcome.
+
-Ran a gel of the reporter system plasmids that Munima isolated yesterday to determine to outcome.
[[Image:Reporter(Oct11).jpg| 250 px]]
[[Image:Reporter(Oct11).jpg| 250 px]]
-
-Set up a Restriction Digest of the Reporter System Parts. pLacI is cut with SpeI and PstI, RFP sub and TetR sub are cut with XbaI and PstI.
+
====Roxanne, Munima====
 +
Objective: Set up a Restriction Digest of the Reporter System Parts and xylE (for metabolic pathway). pLacI is cut with SpeI and PstI, RFP sub and TetR sub are cut with XbaI and PstI.
 +
 
 +
For xylE, TetTsub and RFP sub:
 +
-4 uL of 10x Tango Buffer
 +
-2 uL of XbaI
 +
-2 uL of PstI
 +
-2 uL ddH2O
 +
-30 uL DNA
 +
 
 +
For pLacI and pLacI-3:
 +
-4 uL of 10x Tango Buffer
 +
-2 uL of XbaI
 +
-2 uL of SpeI
 +
-2 uL ddH2O
 +
-30 uL DNA
 +
 
 +
Left in water 37 C water bath overnight.
 +
 
 +
 
 +
===October 12, 2008===
 +
====Roxanne====
 +
 
 +
-Ran a gel of the restriction digested reporter system parts on 1% Agarose in TAE at 100V for 25 minutes.
 +
 
 +
 
 +
===October 13, 2008===
 +
====Munima, Roxanne====
 +
 
 +
Objective: Another restriction digest of reporter system parts, riboswitch and CheZ with XbaI and SpeI.
 +
 
 +
For (DH5a) pzero+CheZ (Spet 30/08), RS3 (Oct. 1/08), and RS6 (Oct. 1/08)[30 uL reaction]:
 +
-3 uL of 10x Tango Buffer
 +
-1 uL XbaI
 +
-1 uL SpeI
 +
-10 uL ddH2O
 +
-15 uL DNA
 +
 
 +
For GFP sub1 [50 uL reaction]:
 +
-5 uL of 10x Tango Buffer
 +
-2 uL XbaI
 +
-2 uL SpeI
 +
-11 uL ddH2O
 +
-30 uL DNA
 +
 
 +
Left in 37 C water bath overnight.
 +
 
 +
 
 +
===October 24, 2008===
 +
====Munima, Christa====
 +
Transformed
 +
- BL21 + pUC19
 +
- RP1616 + pTopp
 +
- GFP complete + RP1616
 +
- DH5a + pUC19
 +
 
 +
====Roxanne====
 +
-Sequencing Results came back in regards to pLacI + (RBS + TetR + dT). pLac i is good, but apparently the (RBS + TetR + dT) sent to us by MIT was not the right part. Reporter system project has been aborted until next year.
 +
 
 +
 
 +
===October 25, 2008===
 +
====Christa & Munima====
 +
Gel extracted pSB1A2 (Xba, Pst) with MiniElute Gel extraction kit. Ran gel after extraction for quantification- 1 uL DNA loaded, but nothing observed; therefore gel extraction was unsuccessful.
 +
 
 +
 
 +
===October 27, 2008===
 +
====Munima & Christa====
 +
Attempted gel extraction of pSB1A7, but no DNA was detected on the gel.

Latest revision as of 02:31, 30 October 2008

Back to The University of Lethbridge Main Notebook

Contents

October 1, 2008

Roxanne

-Inactivated the Enzymes in the morning

-Ran a gel of the tested pLacI, along with the previously cut RFP and TetR in the afternoon on 1% Agarose Gel in TAE.

-the iGEM pLacI had a faint band, RFP looks good (half of it cut), TetR looks like it either didn't cut at all, or only cut once. Only used 2 uL for each sample, will try running with more.


October 4, 2008

Roxanne

-Reran the pLacI tests, RFP and TetR on a 1% Agarose Gel in TAE.

-Repicked pLacI x2, RFP and TetR colonies into LB+amp media since I've been having trouble with ligations. Brent suggested using lots of DNA in the ligation (<100 ng) to make ure that the ligation does in fact work this time.

Argh.jpg


October 5, 2008

Roxanne

-plasmid prepped the pLacI x2, RFP and TetR subcultures. Lost one of the pLacI cultures (cells wouldn't lyse). Had some left over pLacI - 2 culture, attempted to plasmid prep those cells.

-Ran a gel of the plasmid preps.

-Restriction Digest the pLacI - 1, RFP and TetR plasmids obtained today, as well as recutting the TetR plasmid from last time, since it appears as though it didn't cut at all, or only cut once. I will be using the iGEM enzymes. Left to cut overnight at 37.0C

-picked some pLacI colonies from a different plate. Maybe I'll have better luck there. Incubate in LB+amp media at 37.0C for 15 hours.

Oct5gel.jpg


Ocotber 6, 2008

Roxanne

-Running a gel of the parts which were Restriction Digested yesterday.

-Plasmid Prepping the new pLacI subcultures which were incubated overnight.

-Run a gel of the plasmid prep.

Oct6.jpg 2xOct6.jpg

-Yay!!!! The Enzymes Work!!!

Christa, Munima

Made 68 LB + Amp (100 ug/mL) plates and stored them in the iGEM 4 C fridge.

Made 72 5 mL culture tubes of LB liquid media. Stored in the iGEM glass cabinet.


October 10, 2008

Munima

Objective: Isolate more plasmids for continued work on the reporter system.

Plasmid prepped 3 mL of culture for various plasmids for the reporter system: pLacI, TetR sub, pLacI-3, RFP sub, and GFP sub. They are labelled with their name and today's date in 1.5 mL microfuge tubes (50 uL of elution solution). They are stored in the "iGEM Plasmids" box in the -20 C freezer.


October 11, 2008

Roxanne

-Ran a gel of the reporter system plasmids that Munima isolated yesterday to determine to outcome.

Reporter(Oct11).jpg

Roxanne, Munima

Objective: Set up a Restriction Digest of the Reporter System Parts and xylE (for metabolic pathway). pLacI is cut with SpeI and PstI, RFP sub and TetR sub are cut with XbaI and PstI.

For xylE, TetTsub and RFP sub:

-4 uL of 10x Tango Buffer
-2 uL of XbaI
-2 uL of PstI
-2 uL ddH2O
-30 uL DNA

For pLacI and pLacI-3:

-4 uL of 10x Tango Buffer
-2 uL of XbaI
-2 uL of SpeI
-2 uL ddH2O
-30 uL DNA

Left in water 37 C water bath overnight.


October 12, 2008

Roxanne

-Ran a gel of the restriction digested reporter system parts on 1% Agarose in TAE at 100V for 25 minutes.


October 13, 2008

Munima, Roxanne

Objective: Another restriction digest of reporter system parts, riboswitch and CheZ with XbaI and SpeI.

For (DH5a) pzero+CheZ (Spet 30/08), RS3 (Oct. 1/08), and RS6 (Oct. 1/08)[30 uL reaction]:

-3 uL of 10x Tango Buffer
-1 uL XbaI
-1 uL SpeI
-10 uL ddH2O
-15 uL DNA

For GFP sub1 [50 uL reaction]:

-5 uL of 10x Tango Buffer
-2 uL XbaI
-2 uL SpeI
-11 uL ddH2O
-30 uL DNA

Left in 37 C water bath overnight.


October 24, 2008

Munima, Christa

Transformed

- BL21 + pUC19
- RP1616 + pTopp
- GFP complete + RP1616
- DH5a + pUC19

Roxanne

-Sequencing Results came back in regards to pLacI + (RBS + TetR + dT). pLac i is good, but apparently the (RBS + TetR + dT) sent to us by MIT was not the right part. Reporter system project has been aborted until next year.


October 25, 2008

Christa & Munima

Gel extracted pSB1A2 (Xba, Pst) with MiniElute Gel extraction kit. Ran gel after extraction for quantification- 1 uL DNA loaded, but nothing observed; therefore gel extraction was unsuccessful.


October 27, 2008

Munima & Christa

Attempted gel extraction of pSB1A7, but no DNA was detected on the gel.