Berk2010-Amy: Difference between revisions
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===Miniprep Map=== | ===Miniprep Map=== | ||
[[Image:IMG 5443.JPG]] | [[Image:IMG 5443.JPG]] | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Tube''' | |||
| align="center" style="background:#f0f0f0;"|'''Mapping''' | |||
| align="center" style="background:#f0f0f0;"|'''iGEM name''' | |||
| align="center" style="background:#f0f0f0;"|'''Digest''' | |||
| align="center" style="background:#f0f0f0;"|'''Expected length''' | |||
| align="center" style="background:#f0f0f0;"|'''Expected length if still in hybrid''' | |||
| align="center" style="background:#f0f0f0;"|'''Good?''' | |||
|- | |||
| 1,2||2271+2017 w/ PMLL4AC||iGEM10_042||Eco/XhoI||3768, 1672||4702, 1672||? | |||
|- | |||
| 3,4||ig114+2017 g.p. a w/ PMLL4AC||iGEM10_041||Eco/XhoI||3998, 1672||4932, 1672||? | |||
|- | |||
| 5,6||2345 in PMLL4 b w/ 2114+2316 g.p. a||iGEM10_047||Eco/Bam||5533, 2877||||a,b | |||
|- | |||
| 7,8||2345 in PMLL4 b w/ 2114+2275 a (7/14)||iGEM10_048||Eco/Bam||6102, 2877||||a,b* | |||
|- | |||
| 9,10||1968 in PMLL4 b w/ 2114+2316 g.p. a||iGEM10_051||Eco/Bam||5763, 2877||||a,b | |||
|- | |||
| 11,12||1968 in PMLL4 b w/ 2114+2275 a (7/14)||iGEM10_052||Eco/Bam||6332, 2877||||a.b | |||
|- | |||
| 13,14||16a w/ 2294||||Eco/Bam||3166, 2745|| | |||
===Redo of Hybrid vector transfer=== | ===Redo of Hybrid vector transfer=== |
Revision as of 14:39, 20 July 2010
To Do
Tuesday evening:
- Pick colonies off
Background stuff:
- Plan self-lysis timing tests (see Jin's email)
- look up eGFP expression in other single celled eukaryotes. Are we using a GFP that could even express in Choanos?
- look up transposase expression times
Amy N. Kristofferson 13:45, 20 July 2010 (EDT)
None of the colonies picked yesterday from the iGEM10_047, 48, 51, 52, or 16+2294 were co-transformed. And since the ColPCR didn't reveal much of anything, I'm just going to miniprep the first two samples of each, except for iGEM10_047. iGEM10_047's d sample looked great on ColPCR, so I'll miniprep sample a and d instead of a and b. Also, somehow all 12 of the colonies that I picked for the transfer of 2271/ig114+ 2017 from the hybrid into PMLL4AC seemed to still be in the hybrid vector, based on my analytical pcr (they all had bands around 900bp). I'll miniprep four of each anyway and map them. If they're not the right side, I'll do the transfer over again.
To do:
- Minipreps
- Ask Tim about Ef1a sequence.
Miniprep Map
Tube | Mapping | iGEM name | Digest | Expected length | Expected length if still in hybrid | Good? | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1,2 | 2271+2017 w/ PMLL4AC | iGEM10_042 | Eco/XhoI | 3768, 1672 | 4702, 1672 | ? | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3,4 | ig114+2017 g.p. a w/ PMLL4AC | iGEM10_041 | Eco/XhoI | 3998, 1672 | 4932, 1672 | ? | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
5,6 | 2345 in PMLL4 b w/ 2114+2316 g.p. a | iGEM10_047 | Eco/Bam | 5533, 2877 | a,b | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
7,8 | 2345 in PMLL4 b w/ 2114+2275 a (7/14) | iGEM10_048 | Eco/Bam | 6102, 2877 | a,b* | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
9,10 | 1968 in PMLL4 b w/ 2114+2316 g.p. a | iGEM10_051 | Eco/Bam | 5763, 2877 | a,b | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11,12 | 1968 in PMLL4 b w/ 2114+2275 a (7/14) | iGEM10_052 | Eco/Bam | 6332, 2877 | a.b | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
13,14 | 16a w/ 2294 | Eco/Bam | 3166, 2745 |
Redo of Hybrid vector transferThose oligos shouldn't PCR off the genome (http://archaea.ucsc.edu/cgi-bin/hgPcr?hgsid=462798&org=Escherichia+coli+K12&db=eschColi_K12&wp_target=genome&wp_f=cggttatccacagaatcaggg+&wp_r=gcgctgatgtccggcggtgc&Submit=submit&wp_size=8000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0) I'm going to make more of pMLL4AC Eco/Bam digest from Tim's stock in plate 2, well F7 (pMLL4AC Bth8062). Started digestion at 2:04pm. Use 3uL of digested pMLL4AC instead of 1uL when ligating. Unfortunately, there's only about 1.75uL of digest PMLL4AC vector left, so I used it to redo the ligation w/ eco/bam digested ig114+2017. I started the ligation at 1:51pm. Amy N. Kristofferson 14:51, 19 July 2010 (EDT)Everything grew up very well. The only concern I have is that the colonies with 2345 in the part have a transparent loop around the colony, possibly suggesting that self-lysis is starting. I'll have to careful when I miniprep these. I pick colonies, ran a normal colony pcr for all of them, ran an analytical pcr to make sure the parts I transfered out of the hybrid vector aren't still in the hybrid vector, inoculated and check for co-transformation for the assembly plates. Here are my notes:
Analytical PCR results for Transfer from hybrid vectorThis is a gel from a colPCR with oligos th395/th396 of four colonies picked from 2271+2017 and ig114 7/16/10 plates. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. This gel suggests that all the colonies I picked contained the hybrid vector. Tim said that if I need to do the transfer over again, I should use more pMLL vector (~3uL). For now, I'll pick more colonies.
ColPCRThis results aren't very good. But I think that's to be expected when parts are about 6k or greater. I'm going to mostly ignore these results and miniprep a couple samples from each reaction anyway. The analytical PCR makes it seem like all the hybrid transfers are still in the hybrid vector... :(
Amy N. Kristofferson 15:20, 16 July 2010 (EDT)To do: -Eco/Bam digest ig114+2017 g.p. and 2271+2017 g.p. Run on gel and gel purify. Ligate to pMLLAC pre-digested vector. (calculated expected lengths before running gel, to make sure it's worth the trouble. Bands might be so similar that maybe just a zymo is better) -ligate g.p. 2294 with g.p. 16a. -assemble SL-L with vacuole buster:
NOTE: can't use one pot because iGEM10_043 and iGEM10_044 are NOT methylated. I transformed jtk030 cells to make them. I'll have to do separate righty and lefty digests. Sequence Analysis of Ef1a/Atub
So where to go from here? I'll submit atub for sequencing with g00101. As for ef1a, since my ef1a in the AK vector (was supposed to be KA... argh) sequenced perfectly. I'll just Eco/Bam Transfer it? Then I'll get colonies tomorrow, and I can ColPCR/inoculate and then miniprep on Tuesday. OR I could pick more colonies today. However, even if I map well, the part could have mutations... Transfer of 2271/ig114+2017 to PMLLAC from pMLL/1601 hybridEco/Bam digest of 2271+2017 g.p. a, ig114+2017 g.p. a. Gel purify part: 2271+2017 in pMLL/1601 vector Eco/Bam: 3670, 2704 Lower band includes parts. ig114+2017 in pMLL/1601 vector Eco/Bam: 3670, 2934 Lower band includes parts When I pick colonies, I'll run two ColPCRs: ca998/g00101 and th395/th396. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. I'll also run the ColPCR on colonies that I know contain the hybrid so that I can compare. This will allow me to do less minipreps. I'll still map them though, to confirm the vector size.
TH395 gcataGAATTCcggttatccacagaatcaggg fwd for EcoBAM p15A, pBth7011 TH396 ctaggGGATCCgcaccgccggacatcagcgc rev for EcoBam p15A, pBth7011 Second Step of Assembly for SL/VB: L-1968/2345 to iGEM10_043/044Bam/XhoI: 2345 in PMLL4 b, 1968 in PMLL4AC (T1, L) Bgl/XhoI: 2114+2316 g.p. a, 2114+2275 a (7/14) Transposase Assay: ligating gel purified 16a to gel purified 2294 in CORRECT KA vectorDetails of today's Digestions, Ligations, Transformations, and Platings
Submit 2114+2275a 7/13 miniprep for sequencingSubmissions:
Results: Great! This part is as it should be. Sequencing Results for ig114+2017
I'm going to Eco/Bam Transfer the part, because it's currently in a pMLL-jh1601 fusion backbone. Amy N. Kristofferson 14:55, 15 July 2010 (EDT)To do:
Important Mini-meeting update: Issue with SL Assembly probably resulted from use of 2017 in 1601 vectorMapping Yesterday's MiniprepsRan out the gel a bit more, and I think it shows that the two 2271+2017 samples mapped well:
Sequencing Results for the rest of the Minipreps from my first batch of colony picks from Gel-Purified Assembly
The boundaries of parts 2114+2275 and ig114+2017 look good. I'm going to PCR off the middle to check if the insides are go too. I used ca56 for ig114+2017 (binds to pbad promoter). I designed a primer for the middle of Amy N. Kristofferson 13:24, 14 July 2010 (EDT)To do:
Sequencing Submissions: the rest of my gel-purified assembly miniprepsiGEM51-56= each part w/ ca998 and g00101 Cotransformation RedoRescue started at 11;32am. Sequencing Results for 2114+2316 (iGEM10_043): Great news! Successfully put Pcon in front of VBaI submitted the part with ca998 and g00101, and the results clearly show that the Pcon is in front of the vacuole buster. Woo hoo! KA transferChristoph Miniprepped everything he needed. I miniprepped the remaining 1mL of 10 or bjh2294-PMLLKA (eluted with 25uL to keep normal MP concentration), ef1a-PMLLKA b, atub-PMLLKA b,c. Parent Vector bleed-through results: All samples from 1,2,3,4,6,7,8,12,13,16 are good. The only samples with co-transformation were 15a and 9a,b,d.
Colony PCR- ca998+g00101
NOTE: lanes 49-96 refer to the middle gel. Just subtract 48 from the lanes in the table to see what number to refer to on the actual gel. Colony PCR-th313+iGEM10Note: the results for 5 are in the middle right gel shown above.
SL/VB Assembly Colony PicksCotransformation results: 2h and 6a were co-transformed. Everything else had no growth on the triple antibiotics plate.
Amy N. Kristofferson 18:16, 13 July 2010 (EDT)Co-transform jh2348/2291-1601K with Tera's PayloadAdded 100uL of H2O to 1uL of DNA from Tera's Ef1a and Atub payload constructs. Added 1uL of each of those dilutions to 70uL of MG1655 cells. Mg needs to be added for all steps of cloning the PDD's 1M MgSO4 10µL into rescue step (after heatshock) 400µL spread on plate (if +Spec, add 20µL and spread) 30µL into ea mL TB media (use TB for growing up for assay) Started rescue at 4:06pm. KA Vector transfer: ColPCR (parts and vector backbone check), inoculate, grow upI picked colonies, inoculated and ran a colony pcr for all the parts we moved into the new, CORRECT KA vector. Important notes:
SL/VB Assembly: Picking colonies from 7/6, 7/7, and 7/8 platesPicked 8 colonies from Assembly attempt #2 (one pot) and assembly attempt #3 (gel purification) for 2114+2275, ig114+2017 and 2271+2017. Inoculated, checked for co-transformation, and set up ColPCR. Amy N. Kristofferson 14:43, 12 July 2010 (EDT)IMPORTANT UPDATE: TUBE MARKED PMLL6 (KA) in Eco/Bam Box is INCORRECTChristoph just ran and analytical digest/gel that proved that the vector in the tube that was supposed to contain KA actually contained AK vector instead of KA. I need to Eco/Bam transfer the parts into a new KA vector. Also, this explains why the assembly with the Self-Lysis Device didn't work. We need to Eco/Bam transfer it into a KA vector too. Digesting the new pMLL-KA backboneMarianna gave us 10uL of her CMED7 in KA part. The dropout will be 324bp. To digest all of it, I multiplied the Analytical Mapping protocol by 2.5: 10uL of Water 10uL of CMED7-PMLLKA Miniprep 2.5uL NEB 1.25uL EcoRI 1.25uL of BamHI Total: 25uL Load 12.5uL into 2 wells. Gel Purify. Elute each with 12.5uL of H20. Started digest at 4:30pm. Ligation of ef1a and atub (bjh2342) eco/bam zymo'd digestLigated ef1a and atub eco/bam zymo'd digests (from my previous insertion into the supposed KA vector) with the new gel purified Eco/Bam digested KA vector. Started at 7:05pm. Transformed jtk030, MC1061 pir+ cells. In retrospect, I could have used Lefty. Checking vector backbones of SL/VB Eco/Bam transfersSet up a 2K55 PCR reaction with Taq MM, 1uL of 1/10 th313 oligo (in the middle of AmpR) and iGEM10 (rev oligo right before EcoR1 site) to verify the order on antibiotics on the backbone. This reaction PCR's off the AmpR gene to right before the EcoRI site. So if the vector is actually A-, the PCR product will contain a bit of AmpR and plasmid backbone and will be 865bp. If the plasmid is -A, the PCR product will contain a bit of AmpR, lots of plasmid backbone and the other antibiotic. In the case of CA, this amounts to 1808bp. See Eco/Bam page in Excel notebook. This data suggest that all the backbones that should be A- are indeed A- and that all the backbones that are -A are indeed -A. So the antibiotics shouldn't be the problem. For whatever reason, nothing turned up in the 2345 lane. I'll run the PCR again just to double check.
Lane Part Color Expected length Good? 1 Bjh2345a AC 865 no-empty lane 2 Bjh2345b AC 865 good 3 Bjh2345c AC 865 good
Analytical digests of SL/VB Gel Purf. Assembly Minipreps
Payload Delivery: Analytical Mapping of 2348 and 2291 in 1601KBoth parts look great! I'm going to give them to Jin.
Eco/Bam Transfer from Bad KA to Good KA backbone
Amy N. Kristofferson 21:13, 10 July 2010 (EDT)Must do today:
If I have downtime, could start items in the above To Do list From yesterday's colony picks, only the first colony picked from 16a H3 was cotransformed. No idea how that happened, since I gel purified the digests before ligating. Amy N. Kristofferson 13:37, 9 July 2010 (EDT)Colony PCR of SL/VB and 16a+SL AssemblyResults: Very odd. Even though I gel purified the correct bands, it appears that only 2114+2316 worked correctly. I'm going to check to make sure that everything is in the correct vector, because I don't know what else could be causing a problem. Since I'm miniprepping a the couple correct products, might as well miniprep and map a few others to try and figure out what the problem may be.
Animal Promoter: Analytical Mapping of atub and ef1a promoter in pMLLKAStarted an Eco/Bam digest of the 4 minipreps I made of each promoter (all of which had GREAT colony PCRs) at 10:35am. They will be ready to run on a gel at 11:05am. Once it has been verified that these parts map well, I'll submit them for sequencing and then begin the next step of assembly.
Submitted ef1a-a and atub-b Sequencing submission:
Amy N. Kristofferson 14:36, 8 July 2010 (EDT)Cotransformation checkNone of the 2271+2017 or ig114+2017 colonies were cotransformed, but ALL of the iGEM10_018 (16a+SL) were cotransformed. Colony PCR results for 2271+2017, ig114+2017 atub and ef1a promoter, iGEM10_16aCan disregard the results for 16a+SL since all samples were cotransformed. 2017 combo inoculations are useless since nothing showed up on the colony pcr. Very strange, since they weren't co-transformed. GOOD NEWS: atub and ef1a look great.
Redo of SL/VB and 16a+SL digests with Gel PurificationTo avoid co-tranformation and hopefully encourage the creation of the parts I want, I'm going to run a preparative gel, select the appropriate bands and gel purify. Performed a Lefty (Bam/XhoI digest), Righty (Bgl/XhoI digest), and one-pot digest on all the parts in my SL/VB promoter as well as iGEM10_016a and Bjh2294 in PMLL-KA can be found in the Cheshire Chat plate in H3 and F3. I'll gel purify the Lefty digests of Bjh2345, Bjh2271, Bjh1968, Bjh2114, ig114, 16a and the Righty digests of Bjh2316, Bjh2275, and the two self-lysis devices. The other digests are just to make sure everything was methylated correctly and that the one-pot reaction works on these vectors. Notes on protocols: Used Manual 2ab Assembly protocols for Lefty and Righty Mastermixes. Used my One-pot recipe, except with 0.4uL of each enzyme. Also, the DNA taken from the Cheshire Cat Plate was diluted by half b/c it was eluted with 100uL, so we had to adjust the amounts of NEB2 and enzyme used so that the recipe in the R/L digests turned out to be 1.6uL NEB2 and 1.5uL of each enzyme total, since our using 8uL of DNA instead of 4uL of DNA would otherwise have diluted the buffer and enzyme. Digests started at 3:35pm. From left to right: Lefty Digests, Righty Digests Triple Digests Results:
NOTE: lane 2 in the lefty gel is Bjh2294 H3, and everything else is pushed over one lane Summary: Everything is methylated as it should be. I cut out all the right bands and purified them. Also, this shows that triple digestion isn't very efficient and cause a lot of parent vector bleedthrough (the top band of all the lanes). Also, note how dilute the SL DNA is... I'll still elute with the standard 8.5uL to concentrate the DNA, but this is still a potential problem. Ligation and Transformation of SL/VB and 16a+SL purified digestsStarted ligation at 6:45pm. Will transform into jtk030 (pir +, MC1061 cells). I forgot to digest 2017, so I used a righty digest that I had done previously. Started rescue at 8:10pm. Amy N. Kristofferson 13:45, 7 July 2010 (EDT)All the samples from 16a,b + SL were cotransformed, and since Jin mentioned that making a mastermix for the onepot reaction may result in there not being enough enzyme per reaction, I'm going to do it over again without making a mastermix AND using 0.5uL of enzyme instead of o.3uL of enzyme. This should cut back on parent vector bleedthrough as well.
SL/VB Assembly: Redo of One Pot (with more enzyme) for 2114 combos
iGEM10_020 Assembly: redo of 16a+SL assembly, starting with new L/R digests
Possibly successful assemblies (Animal Promoter, SL/VB and iGEM20
Miniprep of Payload Delivery VectorsMiniprepped Payload Delivery stuff (in shaker w/ Mg LB)
Sequencing Results for iGEM10_017b, 2114+2316b,Bjh2345 PMLL4Sequencing results:
Amy N. Kristofferson 13:49, 6 July 2010 (EDT)Colony PCR results for all colonies picked todayNote: they ALL look horrible. Bands in the 16a lanes are probably from cotransformation with 16a vector, since that part is about 850bp.
Transposase Assembly: Colony PCR of iGEM10_016a,b with Self Lysis DevicePicked 4 colonies of each. Ran Colony PCR. Inoculated. Plated to check for co-transformation. Animal Promoter: Colony PCR of Ef1aPicked 2 colonies. Ran Colony PCR. Inoculated. Conor Eco/Bam digested and zymo'd the atub promoter (Bjh2842). Started ligation with PMLL6-KA vector at 4:45pm. Transformed into jtk049 cells. Started rescue at 5:55pm. Payload DeliverySince the payload delivery device contains the Self Lysis device under a promoter that's induced in low levels of Mg, I need to grow up my colonies in LB containing 30mM Mg to prevent Lysis. In the future, I should also rescue/plate on Mg. I selected clear colonies. Tomorrow, I will miniprep the inoculations and map the minipreps next to the parent vector containing the part, so I can make sure the whole part is in the new vector. (Jin said recombination events can occur which could shorten the part) SL/VB Promoter AssemblyRedo one pot reaction with 2271+2071 and ig114+2071Conor set up two one pot reactions and put them in the thermocycler under my ONEPOT program. Added ligase at 4:45pm. Will transform into LEFTY cells. Started rescue at 5:55pm. Picked more colonies for 2114+2275, since miniprep mapping failedPick 8 more colonies from 2114+2275. Ran colony PCR, inoculate, check for cotransformation. Summary of Results so far
For whatever reason, my Colony PCR results and miniprep results don't seem to ever correspond. Amy N. Kristofferson 19:16, 5 July 2010 (EDT)SL/VB Promoter Assembly: Mapping the minipreps produced from Round 1 and 2345 RedoStarted digestion at 5:18pm. Will be ready for the gel at 5:48pm.
Transposase Assembly: Ligation of iGEM10_016a,b Lefty digest with 6+24 Righty digestStarted ligation at 4:05pm. Ligated unmethylated 6+24 digested with BglII+XhoI with both unmethylated iGEM10_016a+b, which had been digested with BamHI+XhoI. Tubes are labelled 16a+SL and 16b+SL (SL meaning Self Lysis, which is part 6). After ligation is over, I will transform into RIGHTY cells. Started rescue at 5:05pm. Plated on CA. Animal Promoter: Ligate Ef1a Eco/Bam digest of PCR product with PMLL6-KAStarted ligation at 4:05pm. Ligated Ef1a Eco/Bam digest of PCR product with pre-digested PMLL6-KA. After ligation is over, I will transform into LEFTY cells. Started rescue at 5:05pm. Plated on KA. Payload: Ligation of Bjh2348 and 2291 with Pjh1601KStarted ligation at 4:05pm. Ligated Eco/Bam digests of Bjh2291 and 2348 with my Eco/Bam digested Pjh1601K plasmid. After ligation is over, I will transform into jtk049 cells, because they don't need to be methylated. Started rescue at 5:05pm. Plated on K. Amy N. Kristofferson 13:00, 2 July 2010 (EDT)Transposase Assay Take 2: Testing time for Transposase expression and time for transposase activity
Unfortunate update: Sequencing proved that iGEM10_020b is a dud. Gibson didn't work because one of our junctions contained palidromic terminal repeats, so the oligos designed based off those junctions didn't have a unique binding site. Oh well. We'll proceed with manual 2ab assembly. iGEM10_20 2ab AssemblyMapping of iGEM10_016 minipreps a and bAn Eco/Bam digest of iGEM10_16 in PMLL5-CK should result in bands at 2662 and 867. Note: iGEM10_017 has already been mapping. (see below) Results were slightly confusing, so I'll submit it for sequencing...? iGEM10_001 was also mapped, and looks good. 1a-c are all good options. Started digestion at 4:55pm. Both have bands at the appropriate places, but 16b has a lot more parent vector remaining, for whatever reason, so 16a is probably the best choice. Assembly of iGEM10_018Lefty Digest of iGEM10_16 Righty Digest of Self Lysis Device (6+24 taken from well G10 of ROMEO plate) Started at 4:42pm. Zymo'd and stored in my working box. SL/VB Promoter Manual Assembly: Analysis of One-Pot reaction2114+2316 and 2114+2275 both successfully transformed righty pir+ cells. The plates are covered in colonies. 2271+2017 and ig114+2071 weren't as successful. The former had no colonies, while the latter had 11, which is surprising, because I used barely any DNA. I will pick four colonies from each, colony pcr, inoculate, and check for cotransformation. Cotransformation results: The ones with letters in the corners were chosen for minipreps.
Moving into jh1601KPerformed an Eco/Bam Digest of jh1601K. Put in incubator at 11:09am. Trashed that digest. Realized I need to do a gel purification, so I'll do the digest again with 4uL of plasmid. Started digest again at 4:54pm. 2345 Redo from 24+PMLL4 digestsThe plate is covered in colonies. I will pick 8 colonies, inoculate, and check for cotransformation.
Amy N. Kristofferson 13:40, 1 July 2010 (EDT)Eco/Bam transfer 2348 and 2291 into 1601KStarted Digest at 12:19pm. Zymo'd and stored in my working box.
2345 Redo: Ligating Tim's ebid 24 and EB pMLL4-ACSince my Colony PCR turned out so horribly yesterday, and 2345 is the Self Lysis Device we've been using for all our construction, I'm just going to use Tim's digests and ligate them together. Started ligation at 11:48am. I transformed the plasmid into lefty pir+ cells. Started rescue around 12:30pm. Plated on AC at 2:13pm. One Pot Assembly of Jin's PartsI'm going to redo the construction of the following with a one pot reaction, since they're all methylated.
Started digest at 11:10am, for all but ig114+2017. I added the speck of 2017 (~.2uL) left at around 11:36am. Started the 20 min heat kill for all but ig114+2017 at 12:30pm. Accidently set the heat kill program for 20 sec instead of minutes and added ligase. Hopefully that won't hurt anything. Put all four tubes in the PCR machine to heat kill at 12:53pm. Added ligase at 1:41pm. Started rescue at 2:42pm.
These numbers have been based on Qiagen minipreps of Jin's pBjh1601
assembly vectors:
1uL lefty plasmid(methylated) 1uL righty plasmid (methylated) 1uL NEB2+ATP 0.3 uL XhoI 0.3 uL BglII 0.3 uL BamHI 6.1uL Water
Notes:
tubes labeled with a red slash
distribute the plasmids into it
1hr at 37 and heat kill steps of the procedure Eco/Bam digest of EF1a PCR productStarted digest at 11:55am. Zymo'd the product. I need to figure out what assembly vector to put it in and then map and sequence the part to make sure it's correct. |