Toronto/Lab Notebook/September

From 2007.igem.org

Latest revision as of 04:02, 27 October 2007

Sept. 30, 2007 (Sunday)

Time: 1:10 PM - 4:23 PM

Esther

Summary:

Done Today:

1. DNA extraction from plates for:

• I13033, E0433, S03140, J04500, J31003 (Note: There may be a problem with this one, check notebook)

2. MP of S01640B

3. Stock cells of S01640B x 4

To Do:

1. Transform items from #1 as well as (J23100+S01640)+B0015 (in freezer).

2. Make LB and plates

3. Recheck (on the computer!) the lengths for PD from Sept. 29th.

• If okay, proceed
• If not, troubleshoot

Sept 29, 2007

Time: 1:20 pm

Anam

• Did PD length check for Q04400 ACD, R0082 #1 & #2, J06501 AB, J13210 AB
  • R0082: cut using X (linearize)
  • Q04400: cut using X/S
  • J13210: cut using X (linearize)
  • J06501: cut using X/S

Recipe:

• 6 μL of ddH₂O
• 2 μL of plasmid
• 1 μL of Buffer 2 (for all)
• 0.5 μL of each enzyme

• Incubated for 40 min in incubator

• NOTE: NO BANDS SHOWED UP FOR Q04400 ACD

Length Check Interpretation:

• R0082 #1 & #2:
  • 1st band - P+P showed up and is correct because this was linearized
  • 2nd band - don't know what this is (it shouldn't have shown up theoretically). It cant be undigested plasmid because it travelled further than the P + P band (1st band).

• J13210 AB:
  • 1st band - May be undigested P + P
  • 2nd band - Correct P + P length because this was linearized
  • 3rd band - don't know what this is (it shouldn't have shown up theoretically).

• J06501 AB:
  • 1st band - may be undigested plasmid
  • 2nd band (showed up only in A) - this is the correct size for the plasmid only but there's no band for the part.
  • NOTE: Part (and plasmid only for B) did not show up for both A & B

• Made 2 tubes of MP o/n and put them in the incubator for S01640 B
  • We only have S01640 A in -80°C freezer stock but since we have S01640 B in the output circuit and will be testing that, we can use that again in the backprop circuit if it works in the output circuit.
  • MP one of them tomorrow and make the other one stock

• Did MP of B0034
  • Found it in the igem box in the -80°C freezer from last year's stock and Charles has said that we can use it.
  • It was labelled "B0034 (2005)" in last year's stock and so I have also labelled "B0034 (2005) MP" on the top of the tube with the MP and put today's date on the side. It is stored in the yellow IGEM box.
  • Do PD length check for this.

OTHER THINGS TO DO AS WELL (Read full lab entry for all tasks):

• Transform I13033, E0433, S03140, J04500, J31003, and (J23100C + S01640B)D + B0015
• Make stock cells and LB(only one tube left...had to throw previous one because something was growing in there) (URGENT)

Sept. 28th, 2007 (Friday)

Time: 4:30pm - 10:00pm

Yusuf, Fareeha

• Finished MP for Q04400 A, C, D
  • Started doing length check for Q04400 A, C, D using enzymes Xba/Pst
  • NO BANDS SHOWED UP FOR ALL THREE
  • COULD BE DUE TO THE FACT THAT WE USED A REALLY OLD GEL SLICE FROM 2 WEEKS AGO BUT THE BANDS FOR THE LADDER SHOWED UP PERFECTLY.. NOTE POINT.. ASK SENIORS ABOUT IT
  • THE COLONIES FROM WHERE Q04400 WAS TAKEN WAS IN THE INCUBATOR FOR 3 DAYS.. NOTE POINT.. ASK SENIORS HOW THAT WORKS

• LIGATED THE PART (J23100 C + S01640B)D WITH TERMINATOR B0015
  • INCUBATION TIME: 1HR 45 MIN AT R.T.P

TO DO FOR TOMORROW:

• Transformation for the ligated part (J23100 C + S01640B)D WITH TERMINATOR B0015
• Try to resolve the part Q04400 controversy do a length check with another enzyme combination
  • If that doesnt work then transform it again

• Length check and Gel extraction and digestion for R0082
  • Just do the length check for R0082 and Q0040 together to save time

• I HAVE MADE A GEL FOR YOU IN THE FREEZER
• CALL CHARLES UP TO COME UP WITH A PLAN AND TELL HIM TO GIVE US A "GO" SIGNAL WITH THE REST OF THE PARTS SO THAT WE CAN START TRANSFORMATION

Sept. 27th, 2007 (Thursday)

Hours: 5 PM - 10 PM

Esther

• Neha
• Rafsan

Results of the MP O/N from yesterday:

• R0082 A, B = successful growth
• Q0440 A, B = no growth

Currently under the assumption that the incorrect antibiotic was added to the O/N for Q0440; retrying once again with KAN resistance.

Note: The antibiotic resistance is written on the plate. Be sure to check it before making an O/N.

1. MP of R0082 A, B
   • Standard procedures used
   • Note: tubes A and B got mixed up, they are now labelled #1 and #2 arbitrarily for differentiation. Colonies of origin are unknown. Re: No longer know which tube came from which colony.
2. Quantitation of (J23100 c + S01640B)D (now referred to in this entry as "insert")
   • Brightness appeared intermediate between fifth band of the 1μL of HindIII ladder and 2μL of HindIII ladder. The lower concentration was used for sake of effectiveness.
   • Chart read 47ng/μg, therefore [4.7ng/μL]
3. O/N of Q0440
   • Colony A: may have had insufficient bacteria. Tried an O/N regardless.
   • Colony B: insufficient bacteria. Did not try an O/N.
   • Colonies C and D: O/N was done with these two, one to replace B and the other in case A was insufficient.
     • Note: You can allow bacteria to regrow at room temperature or incubator if necessary.

Tomorrow:

• Plates must be made (shift started at 5 today, could not autoclave so did not make plates)
• LB must also be made
• Ligate (J2300+S01640) from today with B0015 from yesterday. The former is the insert, the latter is the plasmid. Recipe is in the lab notebook.
• If enough time, transform the above into a plate.

Sept. 26th, 2007 (Wednesday)

Start Time: 3:30 pm - 9:00 pm

Yusuf

• Mini prep o/n from Sept. 24th 2007 is worthless since no body showed up to take it out and finish the MINI PREP. I was still counting on our prior schedule for September.
• SAME Mini prep o/n of (Q04400 -> A & B) & (R0082 -> A & B)
• Doing a digest to do a gel extraction for parts:

      (J23100C + S01640B)D 

• • For detailed info plz check the lab notebook.

• Finished quantitation for B0015
  • Concentration written down in lab notebook and on the eppendorf tube as well.

TO DO FOR TOMORROW:

• Quantitation for (J23100C + S01640B)D
• LIGATION FOR (J23100C + S01640B)D WITH B0015
  • Esther make sure you do the calculation to figure out how much you are going to need properly. If not sure ask Seema or any one of us.
• MAKE SURE YOU FINISH THE MINI PREP O/N WHICH IS IN THE INCUBATOR
• MAKING PLATES, make sure you do it before the wet cycle which is at 3pm exact:
  • 4 AMP + KAN
  • 4 AMP
  • 2 AMP + TET
  • 2 KAN
• Ask Charles about what to do for the new parts that we are transforming and mini prepping.

Sept. 24th, 2007 (Monday)

Start Time: 3:30 pm - 6 pm

Yusuf, Irina

• Mini Prep of J06501 A, J06501 B, J13210 A, J13210 B
• Mini Prep o/n of Q04400 & (R0082 -> A & B)
• Made 4 AMP plates and 4 AMP+CAM plates

TO DO:

• Plasmid digest of (J23100C + S01640B)D
• Then quantitation of (J23100C + S01640B)D and B0015
• Ligation of (J23100C + S01640B)D and B0015

Sept. 21st, 2007 (Friday)

Start Time: 3:00 pm

Yusuf

• Mini Prep o/n for (J23100 C + S01640 B) D.
  • REASONING: Seema suggested that instead of us having the RFP inside of I13507 and since we already checked by PLASMID LENGTH checks and fluorometry that this colony is successful, we could remove it since it sort of staying there is redundant. So all our previous work is sort of redundant as well.

THINGS TO DO FOR TOMMOROW: (ANAM, ESTHER)

• Do mini prep for (J23100 C + S01640 B) D
• Plasmid digest for (J23100 C + S01640 B) D and B0015 which is the terminator in MP form that Seema supplied us with.
• Make the desired plates that we will be needing and we have to confirm with Charles of which we are going to make. The Broth and Agar has been autoclaved and will be on the bench.
• Gel Extract and ligate both (J23100 C + S01640 B) D and B0015
• Possibly do transformations for the new construct that DAN gave us but have to confirm with Charles
• Tidy up notebook and ABBREVIATIONS and also the iGEM boxes in the -20°C freezer.

Sept. 19th, 2007 (Wednesday)

Start Time: 3:30pm - End Time: 9:00pm

Yusuf

• Plasmid Digest of (*L-D + I13507A)A using 22ul of mini prep.
  • There are about 20μl of mini prep left the yellow labeled iGEM box.
• Enzymes used was S/P.

• [[PROBLEM:]] The band that was formed was very thick so it incorporated up to 3 bands in the 1kb ladder.
• The ladders that it incorporated was 5, 6, & 7 even though our expected bp was supposed to be 3093bp.
• We have to keep that in mind for the future if any errors arise.
• The gel has been cut and have been purified and the purified DNA has been put in to the yellow labeled iGEM box.

Sept. 17, 2007

Anam

(Outside lab stuff):

Regarding testing R0011 + F1610A, the following is the basic experimental procedure:

• Transform complete input circuit (R0011 + F1610) into dh5az-1 (done...its in the fridge...length checks have also been done for this)
• Transform complete output circuit (J23100 + S01640 + I13507 + S01003) into dh5a or dh5az-1
• Mix them up and test for CFP production

Theory:

• LuxR genes on output circuit produce luxR protein
• HSL (signal molecule) is produced from luxI gene in the input circuit and pushed out of the cell
• HSL diffuses, binds on to luxI protein and forms complex
• Activates the promoter (R0062) on S01003 in the output circuit and therefore, activates the production of CFP

To Do:

• TO ALL F/T: familiarize yourself with the theoretical aspect of the updated project
• PD, GE, and gel purification (*L-D + I13507A)A (the one that passed the fluorometer test...it has already been mini-prepped)
  • If you don't have time to purify the gel slices, store them in the -20°C freezer and alert the next person who is coming into the lab.
• Quantitate and ligate the S01003(any colony) and (*L-D + I13507A)A together and transform it (dh5a or dh5az-1..it doesnt matter for this circuit but personally i would do it in dh5a just so that when we converse about it, its easy to know which circuit we are referring to [since R0011+F1610A HAVE to be in the dh5az-1])
• PD length check for the complete output circuit after it is transformed (J23100 + S01640 + I13507 + S01003)
• Research excitation and emission wavelengths for cfp
• Test for CFP production

Sept. 16, 2007

Esther

• PD done for S01003 A, B, C, D

• 42 μL of Plasmid
• 6 μL of BSA
• 6 μL of Buffer #2
• 3 μL of XbaI
• 3 μL of PstI
• 3 μL Loading dye
• 63 μL Total

Note on Enzymes: Enzymes are generally not consumed by the reaction. This means that you can digest the amounts of MP used in this lab with 1 uL of enzyme, but you will have to increase the digest time. So the short of it is to try to conserve enzyme when you can by digesting longer. Only increase your quantity of enzyme if you absolutely do not have the time to wait around.

Results:

colonies A, B:

• low yield
• 3 bands: 2000, 1500, 1000

colonies C, D:

• high yield
• 2 bands: 1500, 1000

Gel Extraction:

Weights

• A: 0.22g
• B: 0.23g
• C: 0.35g
• D: 0.31g

Buffer:

• A: 660μL
• B: 690μL
• C: 1050μL
• D: 930μL

• 30μL of TE buffer was used.
• extra allotment of TE buffer has been left out in an eppendorf tube holder. Please use it for the next gel purification.

Sept. 15, 2007

Anam

• Tested for RFP fluorescence using the fluorometer for J23100 + S01640 + I13507
• Only (*L-D + I13507A)A (the red colored ones) were produced RFP

To do:

• PD, GE, and gel purification of S01003 will be done on Sunday by whoever comes in

Sept. 14, 2007

Esther

Note: (J23100 + S01640)= *L

3 M/Ps was done with material from yesterday:

• 1) (*L-B + I13507B)A x 2
• 2) (*L-C + I13507C)A x 2
• 3) (*L-D + I13507A)A x 2 (pink cells)

3 Freezer stocks made with material from yesterday:

• 1) (*L-B + I13507B)A x 2
• 2) (*L-C + I13507C)A x 2
• 3) (*L-D + I13507A)A x 2 (pink cells)

Standard procedure was used for freezer stocks.

3 MPs:

• A standard MP was done.

Plasmid Digest:

• A sample digest was run for all the MP material.

• 2 μL plasmid
• 1 μL BSA
• 1 μL buffer #2
• 0.5 μL PstI
• 0.5 μL XbaI
• 5 μL ddH₂O
• 3 μL 1 kb ladder
• 2 μL loading dye

Results: All results were identical.

3 bands:

• 1) 4000-3500
• 2) 2500-2000
• 3) 2000-1500

Additional Note:

• MP O/N done for:
  • 1) (*L-B + I13507B)A x 2 (possibility of incorrect colony for first O/N. Second O/N done, labelled identically, but with "#2" written in for differentiation).
  • 2) (*L-C + I13507C)
  • 3) (*L-D + I13507A)

Sept. 13, 2007

Esther

• Six test tubes of MP O/N were used from yesterday:
  • 1) (*L-B + I13507B)A x 2
  • 2) (*L-C + I13507C)A x 2
  • 3) (*L-D + I13507A)A x 2

• Note: (J23100 + S01640)= *L

• Today was scheduled to be a fluorometer test for the above items, but there was some question of validity in terms of past data. To be re-examined.

• Therefore: All six tubes were left in the -20°C freezer in eppendorf tubes after aspirating the supernatent.

Sept. 12, 2007

Yusuf

• MP o/n of the following:
  • (*L-B + I13507 B ) A AMP RES = 2 copies
  • (*L-C + I13507 C ) A AMP RES = 2 copies
  • (*L-D + I13507 A ) A AMP RES = 2 copies

Sept 7th, 2007

Morning Session

Yusuf, Neha

Start Time: 11:15am - 2:00pm

• MP of the following ligated parts:

• • (*L-B + I13507 B ) A AMP RES
  • (*L-B + I13507 B ) B AMP RES

• • (*L-C + I13507 C ) A AMP RES
  • (*L-C + I13507 C ) B AMP RES

• • (*L-D + I13507 A ) A AMP RES PINK COLORED CELLS
  • (*L-D + I13507 A ) B AMP RES PINK COLORED CELLS

• • (R0011 + F1610 A) A AMP RES
  • (R0011 + F1610 A) B AMP RES
  • (R0011 + F1610 A) C AMP RES
  • (R0011 + F1610 A) D AMP RES

Evening Session

Yusuf

• After MP I did PD length check for the above MPs. Enzymes X/P was used.
• After talking to Anam, we decided that the bands shown on the gel was acceptable, so we decided to go for MP o/n for the following:

• • (*L-B + I13507 B ) A AMP RES
  • (*L-B + I13507 B ) B AMP RES

• • (*L-C + I13507 C ) A AMP RES
  • (*L-C + I13507 C ) B AMP RES

• • (*L-D + I13507 A ) A AMP RES PINK COLORED CELLS
  • (*L-D + I13507 A ) B AMP RES PINK COLORED CELLS

• I have decided to do two MPs of each: For stock and for gel fluorescence.

Sept 6, 2007

Anam

Start time: 5pm

• 4 MP o/n for R0011 + F1610A (ABCD)
• 2 MP o/n per (J23100C + S01640B) + I13507 plate except for (J23100C + S01640B)A + I13507A because very tiny specks of colonies grew on the sides but the majority of the plate was just fuzzy looking (the plate looked cloudy). One other plate had some fuzziness (cloudy looking agar) in the middle but colonies grew on the sides and looked red (rfp production cuz the circuit works or just some mutation?? stay tuned to find out more).
  • Possible problem with transformation yesterday: Had to throw away a LB tube because something was growing in it at the bottom. We used that tube for the first two transformations protocols that we did. Possibly a hand or something went over the tube when it was open and stuff went in and grew in the LB. The other ligations were incubated using LB from the other tube where (thankfully) nothing grew. This is a possible source of error. Hopefully the PD length check will reveal more.

NOTE: The colonies don't need to look red to the naked eye to know they are working. Seema has used some rfp (I13507) for her own work from a colony that is one of the ones we used and it worked well for her. She also indicated that her colonies didn't to look red, but the rfp showed up nice and bright when tested with the flourometer. So testing will also be performed on the colonies that pass the length check but don't look red to the naked eye.

TO DO:

• PD length check for all so that testing can be done on Saturday.
• After PD length check, make 3 mp o/n from 3 colonies that passed the length check (if those many pass...if there are lesser, just make 1 o/n per colony that passed) and the dilution process can begin on Saturday.

Sept 5, 2007

Evening Session

Start time: 5 pm

Anam, Dan

• Ligated R0011 + F1610
• Transformed ligated parts from yesterday and today
  • All, except one, were transformed in DH5a
  • R0011 + F1610 was transformed in DH5a-z1

TO DO:

• MP o/n for all transformed parts

Sept 4, 2007

Morning Session

Yusuf, Elliott

Start Time: 11:00am

• Making of stock cells:
  • one copy of F1610 (A)
  • Two copies of - J23100 (C) + S0140 (B): All A, B, C, D
    • Overall 8 copies

• Made Plates:
  • 7 AMP Plates
  • 3 AMP and KAN Plates

• Sent pippette tips for autoclaving

• Quantitation for the following GEL EXTRACTS:
  • J23100 (C) + S0140 (B): All A, B, C, D
  • I13507: A, B, C

Evening Session

Anam, Dan, Maria

Start time: 5:30 pm

• Ligation of (J23100 C + S01640 B) + I13507
  • (J23100 C + S01640 B) is labelled as *L - (colony it was picked from)
• Quantitation for F1610 A

TO DO:

• Ligate R0011 to F1610
• Tranform all the ligated parts
• PD, GE, and Gel purification for S01003
• Determine plan for testing input and output circuits and building other circuits (back prop)
• Check if we have DH5a-z1 cells and whether we need to make more (the input circuit needs to be transformed in DH5a-z1)

Sept 3, 2007

Anam, Yusuf

Start time: 12:30 pm

• 1 MP o/n of F1610 A for stock, 1 MP o/n for J23100C + S01640 B (ABCD) (from any colony, they all passed PD length check) for stock
• Use that gel for PD, GE, and Gel purification of I13507 (whichever colonies because all four worked), J23100C + S01640B (ABCD), and F1610 A

To do tomorrow:

• Make AMP plates
• Quantitate J23100 C + S01640 B (ABCD), I13507 ABC, and F1610 A
• Ligate (J23100 C + S01640 B) + I13507 and R0011 + F1610
• Transformation of the ligated parts (if time permits)

Sept 2, 2007

Anam

Start time: 9:30 am

• MP of J23100 C + S01640 B (ABCD)
• PD length checks for F1610 AB (from 2006 plates), J23100 C + S01640 B (ABCD)
  • F1610 AB cut using X/P
  • J23100 C + S01640 B (ABCD) cut using X/P

Legend:

Y - Yes

N - No

To do list:

• make AMP plates
• 1 MP o/n of F1610 A for stock, 1 MP o/n for J23100C + S01640 B (ABCD) (from any colony, they all passed PD length check) for stock
• make gel with big wells by taking the well maker with the big teeth (8 teeth) and use autoclave tape to join two of them
  • Remember to leave spaces between wells when running gel for extraction
  • Determine layout of gel before making the gel
• Use that gel for PD, GE, and Gel purification of I13507 (whichever colonies because all four worked), J23100C + S01640B (ABCD), and F1610 A.
  • Put a total volume of 150-200μL in those big wells

Sept 1, 2007

Anam

Start time: 6 pm

• MP F1610 AB
• MP o/n J23100 C + S01640 B (AB)
  • J23100 A + S01414 A and J23100 B + S01414 B didn't grow on their plates