1. 24 Minipreps for Diezel Blaze in B0034 using Fermentas Kit; stored in -20 freezer
2. Lots of colonies from the transformation last night; no colonies on negation control (good) and lots of colonies on positive control (good) and no satellite colonies (also good); no colonies were picked for overnights
3. Run the Buddy+Benny in B00 and Betty+Enny in B00 Pst/Xba gel from last night at 50V for another 30 minutes for better resolution; all the digest worked so I cut out the appropriate bands and stored the gel in -20 freezer for purification later
For the record:
Buddy = BuOH DH 1235 bp
Betty = Bb-bhBu-coa dh 923 bp
Benny = Butyryl coa dh 1184 bp
enny = Enoyl coa Hydratase 860 bp
Diezel Blaze = buAld-Dh 2639 bp
Moving was done this mid-day.
New location M349
JG has key & access
- MC, ED, JG
Evening crew - please do a once over of G308 to double check we have not left anything and to clean up anything extra/ things we should get out of the way. thnx - MC
Set up new lab
Things we need - Water Bath, Scale, disposable 13ml overnight tubes, Tip waste
Gel purification ran on Betty,enny and boo as well as buddy and benny and is in the -20 in the new lab
To Do list for tomorrow
- Restriction on DB needs to be done - If we want to put it in front of BE or BB then Spe/Pst - If we want to put it behind BE or BB then cut with Xba/Pst - maybe do both (use 4microL DNA for digest)
-complete restriction on BE and BB for SPE and PST (then we can complete ligations BE-BB and BB-BE or we can ligate D.B into BE or BB to make BE-DB or DB-BE or BB-DB or DB-BB- Then we can ligate BE-BB-DB or DB-BE-BB or BE-BB-DB or BB-DB-BE or BB-BE-DB and we are kinda finished!
- If I0500 comes in from calgary it needs to be transformed.
- Transform R0080 which is a back up promoter just in case?
-Note we also have to do sequenceing on BB and BE and DB
Shout out to calgary for saving our necks Shout out to the move in crew to the top
AM Crew Notes:
finished up cleaning up post-move in
got a 'water bath' - JG calibrated it
prepared BB in Boo & BE in Boo for sequencing - this will be ready for pick up tomorrow
- DB in BOO with PST/XBA
- DB in BOO with PST/SPE
- BB in BOO with PST/SPE
- BE in BOO with PST/SPE
NB: there is a section in the masterbook "figuring stuff out" just a few questions I have from being away so long - don't worry about it we should do a wet lab recap to bring everyone on the same page at our meeting Thrusday - MC
<3 JG & MC
Transformed I0500 from Calgary. Transformed R0080. Ran digestions on gel. Ligated digested DB with BB and BE. (all in Boo)
NK ED AL 5:00pm (Unless your crazy enough to show up at 5AM)
Transformed BB Boo DB and BE Boo DB and plated onto Amp plates. (In 37 deg)
Started overnights of R0080 from picked colonies, used Amp. (In shaker)
Restreaked I0500 for single colonies on plates. (Left at 37 deg)
Goodies will be served during the meeting (7:00pm @ CAB 373), so be sure to show up!
AM Crew: MC, JG meeting at 800hrs
No growth observed for R0080 overnights; just scrapping it because we have I0500
No growth on transformations of BB+DB and BE+DB ligations therefore . . . religated - see masterbook for more details
O/N with AMP for I0500
Transform religations in to XL10 Gold
-MC, JG, ML
NB: JG has key access
Mini preps on I0500 overnights
Super Awesome Marathon of Productiveness
DB BE and DB-BB transformations did not work.
Re-ligated DB into BB boo and DB into BE boo
Re-transform BE+DB and BB+DB
Gel extraction of I0500, but we missed the step "restricting with Ecori+speI"
Restrction on I0500 mini's with Ecori and SPeI
And Yet another super productive Sunday
8-11: NG and CZ
NG - Nobody Called me about the key last night (and I completely forgot to swing by: was with parents). So I will wait by the lab either , until the next group shows up or someone with a key. Also bioSci is locked but there is an open door by the physcology wing. I have my cell on 902-8032.
NG - Update: 9:30am 'broke' into lab :D. Then found key...
11-2: VH, CZ
Nick ran the gel of I0500 restricited by Eco/Spe, but nothing showed up as expected (1.5kb).
Digested I0500 and J61003 with Eco/Pst as instructed.
2-5: MC, JG
ran gel of restricted I0500 & J61003; unfortunately there is no DNA in the I0500 sample either
gel extracted J61003
Made LB - to be divided into bottles & autoclaved in G308
O/N of DB in BB & DB in BE samples left in 37degree room
5-8: ML, ED autoclaved little bottles of LB. NOTE: does not need to be autoclaved before you pour it into little bottles and then re-autoclave. Just pour into bottles and autoclave once digested all in Boos except for DB with Pst/xba
Schedule: And Scheduling just got hard core
I have sheduled what we have taken down in the meeting some people appear many times on this schedule working alone just because of the time they were availible. If you are comfortable working alone and taking on lots of shifts im sure the team would be greatful but I'm also sure we would also understand if you didn't want to work alone in the lab. I have only scheduled this way because we need man hours so i tried to fill up all the spots as best i could. -JG
MC,JG for the AM
put away bottled LB
Ran gel of last night's restricted samples "in boo"; did not really turn out
ran gel of uncut & single restricted I0500; uncut I0500 showed 2 bands and restricted showed nothing
Mini prepped Ligations of DB in BB and DB in BE
- run gel of double digested I0500
- do single & double restriction on Mini prep of Ligations
- run gel of uncut, single & double restrictions of mini prepped ligations
ML,AL for the MID (AM crew: What time will you guys be at the lab till? I won't be there until 12:30. Might need to arrange something in order to pick up keys. - AL)
Double digest of BB-DB/BE-DB XBA/PST
NG (2:30-4) for the PM (celine is in calgary if you dont feel comfortable in the lab by yourself I can show up - JG)
Jason if you have time to show up that would be sweet, but if you don't I understand. -NG
Ran gel of the restrictions made in MID, Sept 10 by ML and AL.
JP,ED for the Night
Schedule: No one else really signed up for tuesdays so anyone who has time to drop in should
AM- NK Restrictions of BB+DB with XBA/PST and BE+DB with PST, SPE
Mid - AL (12:30 - 2 ish), NG (random)
Ran gel on restrictions made by NK in AM of Sept 11
Night- ED JP
Ligations of DB+BB + BE and DB+BE + BB
mini relocation 1 bench back, do not use the first 3 benches of the lab or the blackboard from now on!
NG Hi guys I have a meeting 3:30 that cannot be reseached I moved myself to come in Tuesday instead (when nobody was in).
Mid- ML, AL
Important: Please evacuate lab before 2:00pm as there is a lab class starting at this time till 5pm! -AL
PM - VH
lab class 2-5 VH: please make note of the above announcement regarding lab class at 2:00pm
Night - CZ, ED
Transformed BEDBB and BBDB into XL 10 gold
Digested BEBOO and BBBOO with PST and XBA
PM - VH, JG
Ran gel of restrictions made in the previous shift.
After lab shift, JG, JP
Overnights with AMP of BBDB + BEDB
Gel extractions from gel made in the prevous shift
bands 1,2 (BE) correct while band 3 is too large to be BB
Sequence reactions can now be started using primers 1-5 that have been diluted to 5pmol/microlitre in H20 not TE
AM - MC (around 900/930hrs)
- Mini prep from O/N of BBDBE & BEDBB
- Glycerol stocks of BBDBE & BEDBB, and restreaked quartered plates
- Gel extraction of BE & BB Xba/Pst
- made gel
Mid - AL, JG
Restrictions on BBDBE and BEDBB with xba/pst and spe/pst
PM - VH, CZ
Ran gel on restrictions on BBDBE and BEDBB with xba/pst and spe/pst
night - JP
BBDBBE 6 reactions
BEDBBB 6 reactions
BB 3 reactions
BE 3 reactions
General note: Each primer sequence will be the end of the indicated gene to show us wether the joining/ligations worked correctly (VF is the promoter/gene/juction)
To do: Submit to MBSU
And yet another crazy weekend...
8-11 - ED,MC
General Note:We have cloned all 5 genes in series, in to differnt orders. Waiting for I0500 which couls be completed on monday. To make sure we have cloned correctly will also need to wait for the squencing results. This weekdn we will clone the 5 gense in 2 differnt orders. The digest have already been done
Gel extraction of gel bands of XBA,PST of BBDBBE and BEDBBB. THese can be used to cone into I0500 J61003
Ligations of DBBB into BE and DBBE into DBBEBB
Leave at 20 degrees celsius for 10 hours
5-8 - NK Cant find the competent cells.
Competent cells are in -80 freezer 2nd shelf from the bottom-ML, NG
8-11 ED, JG
Transform ligations of DBBEBB and DBBBBE into competent cells.
Plated on AMP with whole ligations
General Info: When DBBEBB and DBBBBE are complete run sequence reaction
Follow "flouresence sequence reaction" protocol
11-2 VH, CZ
2-5 MC, JP
5-8 - ML, NG
No growth on DBBBBE or DBBBBE at 1700
Retransformed into competent cells DBBEBB and DBBBBE
Plated 2 of each and lover overnight at 37 degrees
AM - JG (800 hrs - 930hrs),MC (@ 800hrs)
- delivered MSBU sequencing samples
- O/days of Ligations from yesterday
NB: AMP plates are marked with a red streak down the SIDE of the plate. if it does not have this - then it is NOT AMP
miniprep of DBBEBB and DBBBBE & glycerol stocks
restrict with (1)XBA/PST, (2)SPE/PST
Ruun gel of UNCut DBBEBB and DBBBBE, Cut with XBA/PST and CUT with SPE/PST
GEl extract XBA/PST bands
NK - 8 AM
Sorry guys, I havent done mini-preps before so just incase I mess up i decided not do it. Instead I made a gel for tonight and update the wiki (even the missing parts from before).
VH - (@1pm)
AL - 12-2pm
Mini preped DBEBB and DBBBE, 4 overnights each. Made glycerol stocks of overnights (in -80 deg)
Restrict DBEBB and DBBBE with XBA and PST, and SPE and PST.
if you're the first one in; I0500 arrived - pop by and give Mike a visit!:)
MC - in the AM - unsure what time because has to be downtown for a class project @900hrs (hoping to get out of there within a hour) so maybe 10/1030?
ML - ~10:30 - 1:00
AL - ~1:00 - 2:00
Last Wednesday there was a lab class in our lab from 2-5, is this going to be a regular occurence? -VH
YES - MC
VH, see notes on Sept 12. - AL
Picked up I0500 from Mike and plated on KAN plates.
Made a gel with EtBr in the gel.
Ran a gel of the digests that occurred on September 18th.
Took picture of gel for analysis tomorrow.
NK - 930 AM
VH - (@1PM)
JG, MC- (@5PM)
Picked single colonies from growth on I0500 plates and made overnights and replated on kan plates.
Restrictions on DBBEBB and DBBBE are restarted, XPA and PST, SPE and PST.
Sequencing reactions of Buddy, Betty, Benny, Enny and DB.
AM - MC, JG (@ 800hrs),
ML (~10:30 - 1:00)
AL - ~12:30 - 2:00
ED - 4 PM
Restricted I0500 with ECO and XBA.
Ran a gel of digested I0500.
Took a picture of gel for analysis in the following lab day.
Digested J61003 with ECO and XBA.
Digested I0500 with ECO and SPE.
Made a gel and ran the restrictions described above.
Gel Extracted J61003 band from the gel. (Stored in -20 deg)
Poster Meeting @ 11:00am Meet in sub By the Subway--JG
Repeat digestion of I0500 with 16 microlitres this time.
Ran on a gel, but did not provide a good picture and discarded.
AM -JG @ 8000hrs
MC @ 845 - going to stop by dean of students' to pick up swag first.
PM - NK @5
sorry, cant make it till 630
Started overdays of I0500 picked colonies. (Left in shaker at 37 deg)
Did a mini prep on I0500, followed protocols in Fermentas Kit
1-ish - AL
2pm-ish - VH
TO DO LIST:
looking through the book to last thursday at my sequencing reactions and figure out exactly what samples I used for X-2, X-3, and X-4?
If there is not much left (less than 20 microL), can you transform into bacteria so we can complete another mini? (only 1 transformation for each)
place each of those tubes in a rack or something and leave directions so wayne can find it.
X-1 has two prefixes, so its garbage, but X-5 might be ok. I couldn't find a terminator or a suffix. So... Can you complete a restriction on X-5 with Eco/Spe and Eco/Pst and run on a gel to make sure it cuts properly (and therefore those restriction sites exist)
run sequencing reaction on more X-1 and X-5's (look at the chart erin made from back in the day - its a fold out piece of paper in the master book)
Digest I0500 with ECO and SPE - only half completed because ran out of SPE.
Completed digests from yesterday that could not be finished due to a lack of SPE.
Ran a gel of products of digest but did not yield a good picture and gel was discarded.
VH - 2pm-ish
Redid overnights of I0500 so that they can be Re-minid.
Ran a new gel of the previous minid I0500s to see what is wrong with them. Result shows that there is not enough DNA for these samples to be usefull.
Started PCR for Thiolase.
Made overnights of Buddy in B0034 using the glycerol stocks.
JG & MC - 8:00AM
1-ish - AL
Redid miniprep of I0500 induced by IPTG(1C,1D,2C)and elute in 40 microlitre
Ran 3microliter ona gel but nothing appeared
Miniprep discarded because of lack of DNA
James took 1D culture ands ub culture and perhaps induce with IPTH again.
Restarted 1C and 2C (5ml LB, 5microlitre K, 100microlitre culture)
ED 9:00 am
Made of gel of James's Miniprep digests
Digest I0500 with ECORI and SPE
Transformations of Ligations Enny + J61003 and Betty + J61003
Loaded gel but samples were incorrectly loaded
CZ 2:00 pm
Loaded gel of Erin's digests
Ligation with J61003 E, X
Note:The new TAE is taking much longer to solidify when 1%.
Sequencing reactions of DB in Boo and Buddy in Boo
ED 9:00 Am
Transformed Buddy+J61003, I0500+J61003 #1 and #2
Plated all cells on AMP
O/Ns of Enny+J61003 and Benny+J61003
NG 12:00 am
Tholase blunt end topo reaction
Completed reaction and tholase mix is in orange PCR tube rack at -20
1) We need XGAL!!
2) Colonies that are blue do not have thiolase in them, colonies that are wight do have thiolase in them.