Lab Notebook

From 2007.igem.org

Revision as of 07:06, 29 August 2007

August 27, 2007

Start Time: 11:00 pm – End Time: 3:00 pm

Members Present: Yusuf, Mimi, Talal

• Carried out plasmid digest (length checks) on I 13507 B and J 37033 B

• Length Check results:

I 13507 B – Overall 3 bands were seen for this plasmid

6-7 (Closer to 7 than to 6)

8-9 (Closer to 9 than to 8)

11-12 (Closer to 11 than to 12)

The number of parts and parts and plasmid match up closely but the first band between 6-7 could be of the uncut plasmid

J 37033 B – Overall 2 bands were seen

8-9 (Closer to 9 than to 8)

10-11 (In the middle)

The bands formed doesn’t match up with the given numbers for its part and plasmid from the registry

• Did Mini-prep overnight for F 1610 with 4 colonies labeled A', B', C', D' and put it in the incubator

Start time: 5pm - End time: 9:30 pm

F/T: Anam

- Performed DH5(alpha) + J37033 transformation since none of the PD length checks for all four colonies was odd and the last transformation showed only 5 big, dense circles with very tiny dots around each one even though it was not left for more time than the other transformation. This odd growth might be due to a problem with the transformation. Possible fungus growth? (Seema has informed me that there is a chance that they could grow during the transformation)

August 24, 2007

Time: 5 pm

F/T: Anam

P/T: Talal

-Record the results of the gel Yusuf had made and run (included in table below)

-PD length check for J23100 colony C successful.

-1 band between 6 and 7 on gel

August 23, 2007

Start time: 6:30pm

F/T: Anam

P/T: Fareeha

-Done length check PD for all of A, green circle B, red circle B, black dot B

-Enzymes X and P used for all except for J23100 because the part is too small and so the plasmid that contained it was linearized using enzyme P.

-Buffer 2 was used.

Results:

-All were correct length except for samples from colonies A and B of F1610 and colony A of J37033.

- S01003 (colony A), S01414 (colony B), I13507 (colony A) had 3 bands.

- The 3rd band might have shown up because some of the plasmid was only cut in one location.

To do list:

1) Do a PD length check for the rest of the samples

2) Discuss with Yusuf how much information needs to be recorded when observing the bands in a gel for PD length check

Note: There is one gel left in the fridge. It was made today.

August 22, 2007

Start Time: 5:30 pm – End Time: 7:30 pm Members Present: Yusuf, Anam

• We did Mini Prep overnight and since I did 7 transformations, we decided to do 4 copies of each labeled A,B,C,D so that we have more flexibility in choosing our parts properly. • These are the following symbols we used to clarify all the tubes with the parts

Black circle - I 13507 (AMP Res) (A – D)

Red circle - F 1610 (AMP & KAN Res) (A – D)

Green Dot - S 01640 (AMP Res) (A – D)

Black dot - S 01414 (AMP Res) (A – D)

Red dot - J 37033 (AMP Res) (A – D)

Black line - J 23100 which is a promoter (AMP Res) (A – D)

Red line - S 01003 (AMP Res) (A – D)

• We also labeled our colonies from the Agar plates which were chosen for miniprep overnight as it was instructed by Seema to both Anam and Esther. • The cells which had the promoter J 23100 were light pink colored if Charles and Andy would like to shed some light upon that that would be great.

August 21, 2007

Start Time: 11:00 am

• Charles showed me how to use the registry properly so if anyone needs to know how to use the registry properly just let me know.

o We searched for super parts which is more like a couple of parts ligated together so that it makes our job a lot easier

o So we chose 7 parts to be transformed from the neural network MODIFIED model

• Part timer Muhammad Talal Latif showed up and I instructed him on how to do transformation properly

• So I used 20 l of TE buffer to extract:

o J 37033: 8F, Plate:3 (Amp Res)

o S 01640: 3L, Plate:3 (Amp Res)

• Both of them were from the iGEM 2007 Kit Plates

• We stored the plasmids in the –20C freezer labeled in red DNA remaining

• Added 4 l of J37033 and S01640 to separate competent cell (DH5Z1) eppendorf tubes from the –80C freezer

• So now we are following the Transformation steps from the guidelines posted

• We used two AMP plates made from before

• We also made 8 AMP plates where 4 is going to be used today for the other four parts which we also transformed:

o I 13507: (Used form 2005 stock) (AMP Res)

o S 01003: 20O, Plate:1 (AMP Res)

o J 23100: 21E, Plate: 3 (AMP Res)

o S 01414: 22O, Plate: 1 (AMP Res)

• We also made 3 AMP and KAN plates where 1 is going to be used:

o F 1610: 1B, Plate: 2, (AMP and KAN Resistant)

• CONFUSION: While making the AMP and KAN plates we used AMP of 100mg/ml whereas KAN was 30mg/ml. So even though KAN was of different concentration we used the same quantity.

o CHARLES AND ANDY GIVE US SOME INSIGHT ON TO THIS TOPIC

• So we left all 7 plates for overnight incubation and hopefully we see some results tomorrow

• Everybody please welcome SeungMe (part time) who came in and helped me finish the work today and of course Charles

August 13, 2007

MP and sample PD and gel run.

10 MP total:

5 MP of R0040+E0240 (Black): A, B, C, D, E

5 MP of R0040+E0240 (Red): A, B, C, D, E.

Sample PD was done and gel run.

Enzymes used: X/P

Three bands for all:

~3000, ~2000-2500, ~1000.

Presumably they are undigested plasmid, plasmid alone, and promoter+reporter unit.

August 12, 2007

MP O/N

5 colonies for both petri dishes of R0040+E0240.

Colonies were labelled A, B, C, D, E

Aug. 11/07 ligation dish was labelled in BLACK.

Aug 10/07 (uncertain) ligation dish was labelled in RED.

Tomorrow: MP

Note: DO NOT use the AMP in the iGEM box, as it was left out overnight at room temperature. It may have degraded (meaning, it may be unusable).

In addition:

Eppendorf tubes should be put into Eppendorf tube holding tray, not the test tube holding tray.

Also, make certain you put everything you took out of the fridge back in. Thanks.

August 11, 2007

Ligation + Transformation:

Ligation of R0040 and E0240 done with standard procedures.

Transformation of both today’s ligation and yesterday’s uncertain ligation (marked in RED) for two petri dishes total.

August 10, 2007

PD, GE, and ligation

PD and GE done with standard procedures.

Ligation could have been unsuccessful due to incubation at 37 C instead of rm. Temperature.

Tomorrow:

Religate, transformation.

August 9, 2007

MP and sample PD run on R0040.

Tomorrow:

PD + Gel extraction

Quantitation

Ligation

August 8, 2007

PD of R0040. Mistake made post-gel extraction where all the purified DNA was mixed with loading dye. This mixture is in the –20 freezer at the moment.

MP O/N was prepped with R0040 A from petri dish.

Tomorrow:

Starting at 12 PM:

1. MP (1-2 hrs), make gel while MP.

2. make more agar (while MP)

3. PD test run (1 hr at most)

4. PD + gel purification/extraction (2-3 hrs)

5. quantitation + ligation (2 hrs)

If you’re coming in tomorrow, I recommend you pack lunch.

August 7, 2007

Made stock cells of R0040 and R0011+E0240.

L1/A: 4 tubes

L1/B: 4 tubes

L2/A: 4 tubes

L2/B: 4 tubes

R0040: 4 tubes

They are in the –80 freezer.

August 6, 2007

MP + Test PD

MP: 5 tubes

R0011+E0240 L1 A,B

R0011 +E0240 L2 A,B

R0040

MP was done as usual, except for R0040, 0.5 mL was used to make stock (50/50 mix with glycerol).

PD test run:

Ligated material:

Standard proportions used.

Enzymes: X/P

Total volume: 10mL

R0040:

Standard proportions.

Enzyme: P

Total volume: 10mL

Results:

Expected:

R0040

Part: 54 bp

Plasmid: 2079

Part and Plasmid: 2133

R0011:

Part: 55bp

Plasmid: 2079

Part and Plasmid: 2134

E0240

Part: 879bp

Plasmid: 2079

Part and Plasmid: 2955

R0011+E0240

Uncut: plasmid+promoter+gene 3013

R0040:

One band b/t 2500/2000.

R0011+E0240:

Optimal: Two bands, 2000bp and 1000bp

Possible:

One cut, gene+promote+plasmid

Two cuts, gene+promoter, plasmid

Actual Results:

R0040 adhered to expected results.

Ligated material had anomalies:

L1/A: 1500 bp band = ?

L1/B, L2/A,B: 750 bp band = ? GFP gene alone?

MP O/N done for all five.

August 5, 2007

MP O/N:

Five tubes:

1 x R0040/DH5a (A)

2 x R0011+E0240 L1/(A), (B)

2 x R0011+E0240 L2/(A), (B)

Standard procedures were used. The AMP used is now in the iGEM box.

Future plans:

1. MP for all items.

2. stock tube for E0040.

3. If time allows:

a. Standard PD/gel/quantitation/extraction for R0040.

b. Test PD and gel run for ligated material

i. Use S/P to cut out the insert; if the entire process was successful, the gel should show bands for both the R0011 and the E0240.

Making Stock:

Add same volume of glycerol as in left over MP tube. For this stock, it will be 0.5mL of MP and 0.5mL of glycerol (the glycerol is to protect the cell membranes whilst in the fridge).

August 4, 2007

Did 3 transformations:

1. E0240+R0011 into DH5aZ1, into AMP plates.

2. E0240+R0011 into DH5aZ1, into AMP plates.

3. R0040 into DH5a, into AMP plates.

E0240+R0011 were taken from Aug. 3, 2007 ligated stock.

R0040 directly from iGEM 2007 plate 1, well 70.

For transformations 1 and 2, standard procedures were used with small time variations.

For transformations 3:

1. Extraction from the plate was done using 15mL of elution buffer, not deionized water.

2. Commercial DH5a was used from Seema’s store to increase likelihood of success.

3. Otherwise, standard procedures were used.

Further notes:

1. We require more AMP plates. Whoever is in on Tuesday must make the plates. Remember you need to make the agar right before the autoclave cycle. Time it accordingly.

2. Should we be starting a cloning process with DH5aZ1 if DH5a is successful? Consider uses.

Future Plans:

Sunday: MP o/n prep.

Monday: MP, PD test run + gel test run

Tuesday: Actual PD + gel extraction + quantitate

Wednesday: ligation + transformation

--Esther Kim

August 3, 2007

We did three things today:

1. Plasmid Digest

2. Gel Run + Quantitation + Gel Extraction

3. Ligation

Plasmid and insert (two identical copies each) were run through #1 and #2, and ligated together during #3 (final count: two tubes of ligated material).

Plasmid: iGEM 2006 R0011, colony ?

Insert: iGEM 2006 E0240, colony ? X2

As the materials used were from a successful sample PD from the day before, the entire thing was put through step #2 without a sample PD/Gel run.

Quantitation:

R0011: 9 ng/mL

E0240: 2 ng/mL

Ligation:

R0011 (10 ng): 1mL

E0240 (12 ng): 6mL

10x buffer: 1 mL

ligase: 0.5 mL

ddH2O: 1.5 mL

Plasmid, insert, and water were added first into one Eppendorf tube, then buffer and ligase. Standard procedures were followed after this point.

There were two copies of ligated material.

--Esther Kim