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	<b>Also To Do List (in addition to those listed yesterday): </b><br>		<b>Also To Do List (in addition to those listed yesterday): </b><br>
-	1- restart I0500 PCR + run all 50microL in gel to purify maximum amount -<b>we need this to work!</b> <br>	+	1- restart I0500 PCR + run all 50microL in gel to purify maximum amount -<b>we need this to work because Meagan at MIT hasn't emailed back!</b> <br>
		+	<b> Lab Notes </b>
		+
		+	Ran gel on previous nights digest, 2 triple digest and a -ve control. We also Colony PCR'ed buddy and boo + benny boo and Buddy boo + enny boo as well as 2 I0500. Cut and purified gel of D.B. and then ligated with boo. left on front bench.
		+
		+
		+	NOTE: PCR was left on the machine.
		+
		+	Quote:
		+	"DAMN We are Goood!!!"
	[[Alberta/Calender/August#August|to the top]]		[[Alberta/Calender/August#August|to the top]]
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	<b>Schedule:</b>		<b>Schedule:</b>
-	JP,JG	+	JP,JG,NK <br>
		+	ligations from previous night were not what was expected on gel therefore re-ligated Diezel blaze (xba/pst) with B0034 (speI/Pst. The ligations left at front bench. <br>
		+	ran gel of colony PCR products. Only one of the picked colonies (betty and Enny in boo) had correctly sized insert. The colony could not be matched to the sample, so we started overnights of 4 betty and Enny in boos and 8 benny and buddy in boos.<br>
		+	<b>To do next lab:</b><br>
		+	1)Mini-prep of buddy-benny in boo and betty-enny in boo <br>
		+	2)Restrict 4microL of the mini'd overnights with Xba/Pst - run sample in gel to determine correct insert <br>
		+	3)Transformation of the re-lighated Diezel blaze in B0034<br>
		+	4)PCR of I0500 again. This time use following recipe (as we will need lots!)<br>
		+
		+	-40.5micoL water<br>
		+	-0.5microL taq<br>
		+	-5.0microL 10X buffer<br>
		+	-0.75microL of each primer<br>
		+	-2.5microL of I0500 template<br>
		+
		+	1- Run @ 95 C for 2:00<br>
		+	2- Run @ 95 C for 0:30<br>
		+	3- Run @ 60 C? for 0:30 (check Tm of primers and go  anneal = Tm - 5 C)<br>
		+	4- Run @ 72 C for 2:00<br>
		+	***repeat 2-4 30 times***<br>
		+	5- Run @ 72 C for 5:00<br>
		+
		+	5)WE NEED MORE DNA LADDER!<br>
		+	6)Make chlorobium plates <br>
	[[Alberta/Calender/August#August|to the top]]		[[Alberta/Calender/August#August|to the top]]
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	<b>Schedule:</b>		<b>Schedule:</b>
-	AL,JG,NK<br>	+	AL,JG,NK, NG<br>
		+	NK and JG are meeting up at 3:00 PM, whoever else wants to meet up can meet up then-NK<br><br>
		+	<b>MC available in the AM - because she has to make some phone calls to peoples from the lab - please let me know if you need any work done that can be done within 3hrs via email</b> <br>
-	<b>MC available in the AM - because she has to make some phone calls to peoples from the lab - please let me know if you need any work done that can be done within 3hrs via email</b>	+	<b>AM Lab Notes:</b><br>
		+	Miniprepped all 12 samples; in -20freezer<br>
		+	Transformed religated DB in B0034 into XL10Gold and plated 200ul (x5) of each  - left in 37 incubator <br>
		+
		+	-MC
	<b>To Do List:</b> <br>		<b>To Do List:</b> <br>
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	1 - make Chlorobium plates with and without antibiotics and put them in Capital Health anaerobic chamber<br>		1 - make Chlorobium plates with and without antibiotics and put them in Capital Health anaerobic chamber<br>
		+	<b>Lab Notes:</b>
		+
		+	1-completed mini's of Buddy and Benny in boo and Betty and enny in boo<br>
		+	2-completed restrictions (Xba/Pst) of mini'd samples<br>
		+	3-ran PCR of I0500 <br>
		+	4-ran gel of restricted products and PCR of I0500 - no I0500 - bands for BE and BB may be correct, for better resolution, we ran for longer period after we left last night - NEEDS TO BE CHECKED on friday<br>
		+	5-made Chlorobium plates - 7 amp/7 non-selective and they are in anaerobic chamber (capital health)<br>
		+
		+	-MC,JG,ML,AL,JP, DOUG, NK
	[[Alberta/Calender/August#August|to the top]]		[[Alberta/Calender/August#August|to the top]]
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	<b>Schedule:</b>		<b>Schedule:</b>
-	CZ,JP	+	CZ,JP<br>
-		+	The second gel on the night of aug 30 was not what was expected either. Our Conclusion: The digest likely did not work. However upon Jame's examination of the gel, BB6,7,8 did seem to be right so those bands were cut out and purified for later use.<br>
-	<b>To do list</b>	+
-	1- plate out some chlorobium on selective and non-selective plates (~6:00pm) <br>	+	Therefore we took the following steps:<br>
		+	1. Re-digest mini'd samples->Benny+Buddy Pst/Xbal and Betty+Enny PST/XBal; after running the gel, Betty+Enny4 seemed to work so the band was cut out. It needs to be purified tomorrow.<br>
		+	2.Re-ligate: buddy (xbal/pst)+Benny(spe/PST) and Betty(xbal/PST)+Enny(spe/PST); then transformation of these ligation mixtures<br>
		+	3.Re-transform previousligation mixture :Buddy/enny+Betty/Enny<br>
		+	4. Overnights for Diezel blaze (4ml LB+4microlitreAMP per colony); ready for miniprep tomorrow<br>
		+	Also Note:<br>
		+	We are now using a new batch of XL10 gold competent cells so transformations with Ing pBS(bluescript)as positive control. Same procesdure as before except for our plasmids plate out 200microlitere, but for the positive control, 2 plates of 100 microlitre and 2 plates of 20 microlitre.<br>
		+	<b> For tomorrow, CZ and ML will meet up between 9:30 and 10:00 to do miniprep for the D.B. as well as gel purification of the Betty+ Enny 4 Pst/Xba digest. However, we are not sure if we will have enough time to pick colonies for the Benny+Buddy and Enny+Betty. If anyone could come in to do that, contact either the lab or CZ. </b> <br>
		+
		+	Attendence-NK, CZ, ML, JP
	[[Alberta/Calender/August#August|to the top]]		[[Alberta/Calender/August#August|to the top]]

---

Latest revision as of 03:15, 1 September 2007

Contents 1 August 2 August 1 3 August 2 4 August 3 5 August 4 6 August 5 7 August 6 8 August 7 9 August 8 10 August 9 11 August 10 12 August 11 13 August 12 14 August 13 15 August 14 16 August 15 17 August 16 18 August 17 19 August 18 20 August 19 21 August 20 22 August 21 23 August 22 24 August 23 25 August 24 26 August 25 27 August 26 28 August 27 29 August 28 30 August 29 31 August 30 32 August 31

August

To July 2007
To September 2007
Back to UofA iGEM Home

August 1

Schedule: CZ,NG,VH

General Notes:
1. Run gel of B0034 SpeI/PstI double digest; extract gel with QIAgen kit; stored in freezer.
2. PCR for
a. Enny + B0034 miniprep 1 to 8
b. Betty colonies (10)
c. Buddy colonies (10)
Follow James' protocol. Also see blackboard for James' comment about PCR.

For tomorrow
1. run gel of PCR prodcut which are in the thermocycler �
2. check restreaked single colonies of the Betty and Buddy colony PCR
3. grow up Betty and Buddy overnight and for miniprep and ligation later
4. Make Amp plates (we only have 4 more)
5. Autoclave test tubes

Quotes of the day: We are tired.

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August 2

Modeling Meeting @ 1830hrs in CAB373
General Meeting @ 1900hrs in CAB373
Party afterward's @ Garneau pub

Schedule:

MC, JP & AF are meeting at 1700hrs to get some stuff done before the meeting happens; come join in the fun if you'd like.

Lab Notes:

Made AMP plates
Autoclaved test tubes
Ran gel of ?
Started O/N of Buddy & Betty
Checked restreaked colonies of Buddy & Betty --> positive growth except for 9&10 but this is expected because they are controls

James did a loving PCR of Benny in B0034

-MC, ED, JP, JB

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August 3

Schedule:

Nik cant make it he just isn't crazy enough looks like justin is currently the only one crazy enough
Michelle & Justin are now the long weekend crew - be prepared for ILLUSIONS!!!! or perhaps just some awesome pipetting techniques while they're prepping to champ out at the ULTIMATE pipetting Olympiads.

Meeting @ 1700hrs

Captain's Log, Stardate: 080307-1153

Minipreps of lifeforms Buddy and Betty
Not enough room in box with blue tape, designed a new holding vessel (i.e. box with white tape below box with blue tape in the freezer) for minipreps of aforementioned lifeforms.
-JB

Made gel
Digested (double) Buddy & Betty minipreps
Digest (single) I0500 - from tube we believe is a miniprep in the -20 freezer
weekend to-do list
-JP, ML, MC

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August 4

Meeting at 1000hrs - Michelle will definately be there at 1000 Justin is expected at 1030

Lab Notes:

Made a project timeline - check out the cabinets on the left side of the 4C-degree fridge
Ran gel - no bands for I0500 (we were sad) but there are awesome bands of Betty & Buddy
Attempting to figure out the I0500 problem; transformed into XL10 Gold & HB101

-MC, JP, ML

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August 5

Meeting at 1000hrs - Michelle will definately be there at 1000 Justin is expected at 1030 Lab Notes:

1-transform I0500 into HB101 and XL10 gold
2-restricted mini's of Buddy and Betty with Xba/Pst (confirmed with gel their presence yesterday)
3-ran I0500 samples in gel and no DNA present, chucked out samples - re transform...
-MC, JP & ML (in spirit)

For Tomorrow

1-gel purify Buddy and Betty
2-ligate Buddy and Betty into Boo34 (overnite)
3-start ONs of I0500 for mini on tuesday

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August 6

Meeting at 1000hrs - Michelle will definately be there at 1000 Justin is expected at 1030

Lab Notes:

- JP, ML, AF, MC

Notes:

1- re-plated I0500 tranformations on Kan and AMP plates - hope for growth
2- ran buddy and betty XbaI/PstI restriction products on gel and gel extracted
3- began ligation reaction with B0034 (in back shaker at 13 degrees)
4- started making butanol plates...but it might not work so well with higher percentages because when you plate the agar its warm/hot and when you add the butanol it bubbles like mad (evaporates). We tried pouring at lower temp, but it prooved disasterous

Fact of the Day

A good ligation is like a hoola-hoop, they're both circular and only one is methylated

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August 7

Schedule:

MC,VH,AF

Protocol for Today

1: If there is growth on I0500 plates in 37degrees, make some overnites of 5 colonies (4ml LB + 2.4microL KAN [in buffer box]in shaker tubes and innoculate with bacterial colony and place in shaker at 37degrees overnite -circle each colony you pick and match with respectively labeled shaker tube)
2: Take the two ligation mixtures from the back room shaker- transform into XL10Gold bacteria- for each of the sample make two dilutions.
dilute (1)2microL into 8microL water and
(2)1microL into 9microL water.
Take the four samples and transform them (dilute in 600microL LB before puting into shaker.
Plate each transformant onto AMP plates in two dilutions:
(1)200microL on a plate
(2)50microL + 50microL LB.

Lab Notes:

No growth on Kan plates of I0500 in HB101 - therefore no O/N
Transformations of Buddy+B0034 Ligations & Betty+B0034 Ligations into XL10GOLD competent cells
- Betty+BOO34 10x = 1ul Lig + 9ul H20 (the number on the side of the eppendorf denotes the ratio of vector to insert in the lab book) 20x = 2ul Lig + 8ul H20
- labelling same for Buddy+BOO34

- MC, VH, AF

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August 8

Today is sweatpants day. Wear your favorite pair.

The last of the synthetic genes has arrived (butyraldehyde dehydrogenase = BuAld_DH). I resuspended the plasmid in 40uL TE and put the red capped tube (with a pink tape flag) in the freezer in G308 - MKD

LAB NOTES:

1: transformed buAld_DH "Diezel Blaze" into XL10 gold need to grow up for mini tomorrow nite
2: colony PCR on buddy and betty in Boo- reactions are in PCR machine (hopefully at 4 degreesC)
3: started Chlorobium medium/concoction need some more compounds

-JP,ED,AF

Schedule:

ED,NK,JP

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August 9

Schedule:

ED,AF,JG

Lab Notes: -poured 1%-4% butanol-LB plates - added butanol to each individual plate as it was cooling. plates are on bench #3

-ran gel of PCR of Buddy and Betty in Boo. Looks like all colonies tested were positive. Started overnights of positive colonies for glycerol stock and miniprep tomorrow.

-set up PCR using VF and VR and 0.5 uL of 1/10 dilution of I0500 stock as template. Mg titration of 1,2,3 mM. Need to be run on gel tomorrow

-started overnights of Diezel Blaze for miniprep and glycerol stocks

-ED,MC,JG

Modeling meeting cancelled as Nick G. and Doug are both away - WM

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August 10

Schedule:

CZ, ML, JG
Thanks for taking my shift ML - MC
Please note that if you cannot make a shift we have already discussed that you are to call the lab phone number which is in previous minutes - I can send it out with the next minutes just in case. - MC&JG

Lab Notes:

Minipreps and glycerol stocks of Buddy and Betty in Boo and Diezel Blaze were done and stored in box with green tape.

Tonight: run I0500 PCRs on gel, digest Diezel Blaze to clone into Boo, start mutagenesis experiments, transform some new promoters

This weekend: we can start cloning our genes into J61003 with this promoter and then swap the promoters later if we want (this was the reason for picking J61003), also we need to wash and autoclave culture tubes

-ED

Lab Notes

Ran gel of pcr product no bands where observed. PCR reminants are stored in -20 freezer.

Double digest of diesel blaze pst1 xba1, Stored in -20 freezer

Gel already prepared on the second bench.

Culture tubes have been wash but need to be autoclaved tommorrow

ACTION ITEM: decide on promoter to transform we promoter list is on the mac computer.

Uplifting thought:

A negative result is still a useful result. As long as it is within errors it is still valid.

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August 11

Schedule: 9:00am sharp as a needle

JP, AL, NK

1. got a hold of Meagan at MIT and she's sending a stub of IO500 bacteria. we should get it early next week I hope!

Lab Notes:

1- organized all important samples into new box in -20 (all coding sequences in Boo) These will need to be restricted with Xba/PST for ligation into J61003 later
2-started ligation on Diezel into Boo (samples at the front of the room on bench - need to transform tomorrow asap)
3- autoclaved

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August 12

Schedule:

CZ, AL, NK

Celine: wanna meet up at noon? AL

Lab Notes There was a misunderstanding about the Diezel Blade double digest. On Friday, we only did the digest but did not run a gel or perform gel extraction. So the ligation mixture from Saturday was discarded because B0034 was ligated to the double digest mixture directly.

Today we ran the remaining double digest mixture; however it did not cut.

Also we transformed Bba_I13453 (pBAD promoter by itself) just in case on Amp plates, which are in 37 degree incubator.

For tomorrow 1. Take out I13453 plates and put in 4 degree fridge (Celine will do this early morning around 8am) 2. Pick colonies for I13453 and grow in Amp-LB overnight 3. Double-digest of Diezel, run gel, gel extraction, ligation with B0034.

Justin, can I swap with you for Monday because I may not make it on Thursday if we are doing labwork before the meeting? Email me to confirm. Thanks. - CZ

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August 13

Schedule:

ML,JG,JP

Omniscient Notes:

1- Label Tubes Well - There is nothing worse than having to redo your samples because you don't know what each tube is. Aside of our coding namings Benny, betty etc, Label in detail. Always put the date on the tubes so we can always refer to the master book if we're confused

2-We need to be making better lab notes in the notebook -

"Started Overnights of..." is not acceptable. Instead, write what you added to the tube: 5uL Amp, 5mL LB, colony.

Likewise, "Double digest" is not acceptable. You need to write down what you digested and what amounts of everything has been added.

Writing in a notebook correctly is very important with this many people in the lab. If people are forgetting to add buffer to digest or antibiotics to overnights, this needs to be corrected immediately and by reading what was done in the lab book, it can be. It is much better to write too much than too little, especially if it can jeopardize the future of the project.

I know that I have been guilty of this, but we all need to improve. Everything needs to be written down.

Lab Notes:

-I took out plates for I13453 this morning and there are big colonies to pick tonight.
-Started overdays of Diezel Blaze and I13453.
-Digested D.B with Xba and Pst.
-Poured gel for use tonight.

-MC, ED

Tonight:

-Miniprep and digest I13453 with EcoR1 and Xba tonight. Miniprep and store D.B in freezer tonight (digest with XbaI/PstI).
-made glycerol stocks of each D.B and I13453

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August 14

Schedule: MC,JP,ML

To Do:

1-run gel of D.B restriction products
2-gel isolate D.B (XbaI/PstI)
3-start ligation of D.B in Boo

Things We're Feeling - a monologue by Justin & Michelle (& Matt - he sends his apologies):

Ran a gel for 2 hours to try and resolve D.B (2650bp) and pUC17 (2700bp); don't laugh
Diezel Blaze is actually kind of the same size as the plasmid it came in (PUC17)

there were tears

some precision cutting happened - we're going to chance it
we hope that most of what was cut out will have mostly Diezel Blaze in it - however to compensate when Ligations are done B0034(vector) should be added in EXCESS to increase chances of B0034 + Diezel Blaze = <3

we'll have to do some colony PCR with VF/VR after transformation and the rxn's that work will be BOO34 and the ones with inserts the right size will be the correct configuration. We'll definately need to sequence...

end scene

For Tomorrow:
- gel extraction of D.B (in new box in -20)
- Ligation with excess B0034 ( 2microL Boo + 2microL D.B. and 2microL Boo + 4microL D.B.)
- expecting bacterial stub of I0050 in the mail if it is in - then grow it up

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August 15

Schedule: CZ,AL,NK

Lab notes:

Gel extract Diezel Blaze using QIAgen Kit.

Ligated product with BOO34. (1:2 and 1:1 vector: insert ratio)

Also James dropped by and was wondering about sequencing. The sequencing reaction protocol is at the front counter and takes about 2 hours, perhaps we can do it on this weekend for all of the plasmids we have so far.

Where are all the plasmid maps for the genes just in case we need to find it?

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August 16

Schedule:

JP,CZ,AL If lab can happen before meeting we can just leave for erins house after the meeting so you three should look into that JG

I most likely cannot meet at the lab before the meeting, but if either Erin or Justin can. I would be willing to swap. Please let me know ASAP, thanks.-CZ

I can go in before. - ED

What time are we meeting up for the lab? -AL

Post-meeting video/board game party at Erin's house if there is interest.

Lab Notes:

Transformed ligations into XL10s and plated on Amp. Plated 50uL and then spun down the remainder of the tube and plated that.

Tomorrow:

Colony PCR of Diezel Blaze in Boo and find out what is going on with I0500.

-ED

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August 17

NB: Meeting for ANAEROBIC LAB tour at 1450hrs at the University of Alberta Hospital "starbucks" across from the gift shop

Schedule:

ED, AF, JG

Morning Lab Party:

Sorry Jason - I did not see you were available until now . . . . .
Colony PCR (14 samples) from the Diezel Blaze in B0034 transformations
- PCR samples will be in the left Thermocycler
- Restreaked plates of PCR samples in the 37 degree incubator
- Put the transformation plates in the 4 degree fridge in the back without parafilm
-MC

Evening Lab Notes:

Poured Amp plates.
Digested BuddyB BennyB BettyB EnnyB (B=Boo) with Xba.
Ran gel of colony PCRs of Diezel Blaze in Boo.
Got two differend band sizes, one at about 2700 and one about 3600.
Will grow up both so they can be submitted for sequencing

-ED,CZ,JG

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August 18

Lab marathon this weekend! Start @ 10:00 and goes all day. Everyone show up. We will be leaving mid-afternoon and returning at 8:00 PM.

I'll be there at 1100ish - AL, jp

Schedule:

ED, AL, NK

Lab Notes:
Continued with digest from Aug17 & Ran gel - results were different from expected
Overdays of Diezel Blaze in Boo34
Repeated Digests & ran gel again - unfortunately results were different from expected AND previous gel's results
Glycerol stocks of Diezel Blaze in B0034
Mini Prep Diezel Blaze in Boo34

- MC, ED, JP, AL, ML

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August 19

Schedule:

CZ, AL, NK

Lab Marathon Continues

Meet at noon for more marathon goodness.

Oops on FAVA!!!!! They have given away the camera the Anthony had reserved so there will be no movie this weekend :(

Lab Notes:
Sequencing of all genes (BennyB, BuddyB, BettyB, DiezelBlazeB, EnnyB) in B0034 - only did two of each in the case where there were more than two mini preps
stored at -20freezer taped together labled "to MSBU" - will be delivered to MSBU tomorrow

-MC, JG, CZ

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August 20

Schedule:

ED,JP,JG

Digested all "in Boos" for further confirmations, but failed to restrict DB #2 and #7.

Prepared chemicals for anaerobic broth.

Childrens Story of the Day

"Paddy cake, paddy cake, bakers men, bake me a cake as fast as you can"

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August 21

Schedule:

AF,JP,ED

Lab Notes:

1-ran gels of re-digested "in Boo's" for further confirmation. Realized that D.B samples 2 + 7 should have been restricted too!
2-confirmed sequencing results - Buddy and D.B. need to be re sequenced - D.B. 2+7 along with Buddy samples (started overnites tonight - mini tomorrow - digest/sequence) All others are grandioso

3-Started overnights of Buddy in boos which will need to be restricted with Eco/speI (run for size confirmation -but KEEP nicely labeled samples for isolation of RBS-Buddy for puting in J61003)

4-email Meagan from MIT again to see where our bacteria is!

5- make trace elements for Chlorobium culture

Childrens Story of the Day

"Paddy cake, paddy cake, bakers (WO-)men, bake me a cake as fast as you can"

-ED, JP

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August 22

Schedule:

ED, AL, MC

General Notes:

1- Dr. Foght was able to give us 25ml of 0.1mg/mL Resazurin she also gave us CuCl2 2H20, FeCl2 4H20, NaSe03 (to replace NaHSeO3- careful its poisonous!), VoSO4 2H20!!! We got more than we barained for!!! Thanks Dr. Foght! - jp

Lab Notes:

Miniprepped Buddy overnight (Buddy in B0034), stored in -20 degree freezer.

Sequencing prep for Diezel Blaze #2&7.

PCR I0500, ran on colony PCR in right thermocycle. (Need to be run on gel tomorrow, in PCR machine on the right.)

Digested all with Pst; Buddy and Betty with Xba; Benny and Enny with Spe. Samples put in freezer, to be run on gel tomorrow.

Updated cloning progress in lab book.

2 gels poured for tomorrow.

Trace elements, salt A, B and C for the Clostridium made: dissolved in distilled water and autoclaved. Left on bench to be cooled. Trace elements are at 0.5 X'

-ED, MC, AL, AF

Friendly tip of the day: "Always wear gloves when handling EtBr! "

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August 23

Schedule:

Performed Gel Extraction on I0500 gel August 23rd AM

Cut out bands for Buddy restricted with Xba and Pst and Betty restricted with Xba and Pst from August 23rd AM gel 2.

The Benny and Enny restrictions DID NOT work.

Chlorobium medium is complete and ready for innoculation at 1700 August 24th.

Emailed Meagan at MIT to ask for I0500 again (incase our PCR'ing doesn't work)

In attendance:

ML, JP, AF, JG, AL

Things for tommorow/weekend:

1- Restrict I0500 samples (See July 14th 07 for protocol). (both tubes are the same sample) with EcoR1 and Xba.
1a- sample 1 - 5microL I0500 DNA + 1microL T4 ligase + 4microL 5X ligase buffer + 10microL ddH20
1a- sample 2 - 15microL I0500 DNA + 1microL T4 ligase + 4microL 5X ligase buffer + NO WATER

2- Restrict Benny and Enny in Boo's (in new unlabeled IGEM box-with other three boxes (DNA DNA and Buffers...) (with PstI/SpeI- there should be a protocol in master book within last week)

• use 4microL "in boo" DNA of each sample

3-Run Gel, Purify, Ligate I0500 into J61003 (that has already been restricted with EcoR1 and Xba), (freeze Benny and Enny gel purified SpeI/PstI)
4-Transform I0500 in J61003 into comp Hb101, sequence... rxn

1.Extract the bands that have been cut out (are next to I0500 in box); Betty and Buddy from Aug 23rd AM gel,.

Sequencing of D.B in B00 #2 and #7 has been done. I will update the results Friday evening. -ED

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August 24

Schedule:

ML, JG, NK
Thanks justin-NK

LAB WORK

Restrictions where done on I0500 and Enny in boo and Benny in Boo, No I0500 bands where found on the gel, Bands where seen for benny-boo and enny-boo However gel got mangled in the process of moving it from camera to lab so no gel purification was not done.

S.T.D. (Stuff to do) The restriction on Enny in Boo and Benny in Boo needs to be done. Gel needs to be reran and gel purification needs to be done. So pretty much everything that we did tonight lol.

Quote of the Day

"I became president to lead not to read!!!"

Attendance NK, JG, AF, ML PEACE!

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August 25

Schedule:

CZ, NK, MC
If you are having trouble mangling gels, you should carry them to the camera inside of the tray that you pour them in.

Lab Notes
The I0050 sample on gel from august 24th is not new, it is the PCR sample
It did not show on the gel
Who purified Buddy and betty?

Lab work
PST/Spe restriction of Benny+boo (1,3)
PST/Spe restriction of Enny+boo (6,9)
Used 4 microlitre of sample
Ran gel at 100V
Dishwashed and LAB clean up, ethanoled all benches we usually work on

In attendance: NK, MC, CZ

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August 26

Schedule:

CZ, AL, NK
Lab work
1. Gel extraction; labelled as Benny in Boo (1) pst/spe gel purified
Benny in Boo (3) pst/spe gel purified
Enny in Boo (6) pst/spe gel purified
Enny in Boo (9) pst/spe gel purified

2. ligation
a. Buddy in Boo pst/xba + Benny in Boo (1) pst/spe
b. Buddy in Boo pst/xba + Benny in Boo (3) pst/spe
c. Betty in Boo pst/xba + Enny in Boo (6) pst/spe
d. Betty in Boo pst/xba + Enny in Boo (9) pst/spe

Store at the front counter overnight for transformation tomorrow.

In attendance: AL, CZ

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August 27

Schedule:

CZ,JP,NK
hey guys, iv got a meeting at 6:30, so i might be a little late-NK

General Notes:

1. after comparing the sequenced Diezel Blaze samples (1,2,4,7) with Erin's sent out sequences, the construct is not correct. Therefore tonight we must complete restriction of D.B with XbaI/PstI and re-ligate it in Boo that has been restricted with XbaI/PstI.

Lab Notes
1. After talking to James, we decided to do a triple digest (Pst/Xba/SspI). SspI is an unique site inside the D.B. vector (i.e.pUC57) but absent inside D.B. Ssp works in Buffer O. So Pst/SspI digest in Buffer O for 1.5 hours at 37 degree, then Xba in Buffer Tango for 1 hour. Also we did a negative control where D.B. is digested with SspI only.
Both tubes are stored in a box with white tape in the -20 freezer. Gel is already pour for tomorrow.
2. Transformation of yesterday's ligation (Buddy+Benny, Betty+Enny) into DH5alpha and stored in 37 degree incubator overnight.
3. Appears to be some Chlorobium growth - MEDIUM WORKS! - the next step is to make plates and see if they work - making the plates will happen on thursday
For Tomorrow 1. Run gel of tonight's digest. For the negative control, one should expect a band around 5.3kb(unique site, linearized vector 2.7kb + D.B. 2.6kb). For the triple digest, one should expect the D.B. band at 2.7kb and the vector band at 2.0kb. The upper 2.7 kb is the one to be cut out, gel purified, and ligate with Boo like previously overnight.
2. If there are colonies from tonight's transformation, colony PCR and run gel if they are ligated together properly.
In attendance:
JP, CZ, NK

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August 28

Schedule:

I will drop by briefly to see the gels and the colonies out of curiosity. -CZ,JP
AL,JG

Also To Do List (in addition to those listed yesterday):

1- restart I0500 PCR + run all 50microL in gel to purify maximum amount -we need this to work because Meagan at MIT hasn't emailed back!

Lab Notes

Ran gel on previous nights digest, 2 triple digest and a -ve control. We also Colony PCR'ed buddy and boo + benny boo and Buddy boo + enny boo as well as 2 I0500. Cut and purified gel of D.B. and then ligated with boo. left on front bench.

NOTE: PCR was left on the machine.

Quote: "DAMN We are Goood!!!" to the top

August 29

Schedule:

JP,JG,NK
ligations from previous night were not what was expected on gel therefore re-ligated Diezel blaze (xba/pst) with B0034 (speI/Pst. The ligations left at front bench.
ran gel of colony PCR products. Only one of the picked colonies (betty and Enny in boo) had correctly sized insert. The colony could not be matched to the sample, so we started overnights of 4 betty and Enny in boos and 8 benny and buddy in boos.
To do next lab:
1)Mini-prep of buddy-benny in boo and betty-enny in boo
2)Restrict 4microL of the mini'd overnights with Xba/Pst - run sample in gel to determine correct insert
3)Transformation of the re-lighated Diezel blaze in B0034
4)PCR of I0500 again. This time use following recipe (as we will need lots!)

-40.5micoL water
-0.5microL taq
-5.0microL 10X buffer
-0.75microL of each primer
-2.5microL of I0500 template

1- Run @ 95 C for 2:00
2- Run @ 95 C for 0:30
3- Run @ 60 C? for 0:30 (check Tm of primers and go anneal = Tm - 5 C)
4- Run @ 72 C for 2:00

• • • repeat 2-4 30 times***
      

5- Run @ 72 C for 5:00

5)WE NEED MORE DNA LADDER!
6)Make chlorobium plates

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August 30

Schedule:

AL,JG,NK, NG
NK and JG are meeting up at 3:00 PM, whoever else wants to meet up can meet up then-NK

MC available in the AM - because she has to make some phone calls to peoples from the lab - please let me know if you need any work done that can be done within 3hrs via email

AM Lab Notes:
Miniprepped all 12 samples; in -20freezer
Transformed religated DB in B0034 into XL10Gold and plated 200ul (x5) of each - left in 37 incubator

-MC

To Do List:

1 - make Chlorobium plates with and without antibiotics and put them in Capital Health anaerobic chamber

Lab Notes:

1-completed mini's of Buddy and Benny in boo and Betty and enny in boo
2-completed restrictions (Xba/Pst) of mini'd samples
3-ran PCR of I0500
4-ran gel of restricted products and PCR of I0500 - no I0500 - bands for BE and BB may be correct, for better resolution, we ran for longer period after we left last night - NEEDS TO BE CHECKED on friday
5-made Chlorobium plates - 7 amp/7 non-selective and they are in anaerobic chamber (capital health)

-MC,JG,ML,AL,JP, DOUG, NK to the top

August 31

Schedule:

CZ,JP
The second gel on the night of aug 30 was not what was expected either. Our Conclusion: The digest likely did not work. However upon Jame's examination of the gel, BB6,7,8 did seem to be right so those bands were cut out and purified for later use.

Therefore we took the following steps:
1. Re-digest mini'd samples->Benny+Buddy Pst/Xbal and Betty+Enny PST/XBal; after running the gel, Betty+Enny4 seemed to work so the band was cut out. It needs to be purified tomorrow.
2.Re-ligate: buddy (xbal/pst)+Benny(spe/PST) and Betty(xbal/PST)+Enny(spe/PST); then transformation of these ligation mixtures
3.Re-transform previousligation mixture :Buddy/enny+Betty/Enny
4. Overnights for Diezel blaze (4ml LB+4microlitreAMP per colony); ready for miniprep tomorrow
Also Note:
We are now using a new batch of XL10 gold competent cells so transformations with Ing pBS(bluescript)as positive control. Same procesdure as before except for our plasmids plate out 200microlitere, but for the positive control, 2 plates of 100 microlitre and 2 plates of 20 microlitre.

For tomorrow, CZ and ML will meet up between 9:30 and 10:00 to do miniprep for the D.B. as well as gel purification of the Betty+ Enny 4 Pst/Xba digest. However, we are not sure if we will have enough time to pick colonies for the Benny+Buddy and Enny+Betty. If anyone could come in to do that, contact either the lab or CZ.

Attendence-NK, CZ, ML, JP

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