Team:DTU-Denmark-2/Notebook/Lab notes

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Lab notes

July



Week 1: 04.07.2011 - 10.07.2011

Week 2: 11.07.2011 - 17.07.2011

Wednesday

First primers have been ordered, so we are soon ready for lab.

Week 3: 18.07.2011 - 24.07.2011

Tuesday

First day in the lab and 48 biobricks to go, how exciting!
Of the 48 biobricks we will have when the project is finished we had only established 17 of them and ordered primers. We started the day with 11 PCR reactions of the first bash of 17 biobricks, we still need some DNA templates to due all 17. 6 PCR were correct and was purified with a purification Kit (GE Healthcare). So only 42 biobricks to go!

Biobricks in the bag:
  • pALC
  • TtrpC
  • Terminator 1
  • Terminator 2
  • Terminator 3
  • argB


Thursday

Some of the PCR reactions that didn't work Tuesday were repeated with a different annealing temperature and amplification of new biobricks.

New biobricks in the bag:
  • PGK
  • BGHpA
  • eGFP+k_GOI


The fragments will be frozen and purified later.

We still have not succeed to make biobricks pyrG and pyrG-DR (use as a marker in fungi), despite two PCR attempts with a high annealing (59°) and low (56°) temperature.

Work for iGEM Copenhagen team
We have established collaboration with iGEM Copenhagen team, where we will create some biobricks for them and they can use our plug 'n' play system and they will save a lot of time. So today we transformed competent E.coli cells with some of the standard biological parts from iGEM, BBa_R0010, Bba_B0015 and BBa_J52034, so we later take make biobricks to match our system.

Friday

The biobrick which we still have not succeed in making with PCR is tried again with small modification. We did the PCR reaction with and without MgCl2 50mM in the reactions.

Biobrick obtained:
  • CMV
  • hygR


The fragments will be frozen and purified later.

Week 4: 25.07.2011 - 31.07.2011


Monday

We got a new batch of primers today. They need to be diluted and stocked, before they are ready for use. We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.

The PCR reactions were successful, so following biobricks are ready for Plug'n'Play:
  • SV40 +ori
  • SV40 pA
  • LacZ
The fragments will be frozen and purified later. We still have trouble with obtaining pyrG and pyrG-DR.

Tuesday

The new primer batch we recieved yesterday, will be used for amplification of many many many new biobricks today.

Out of 15 PCR reactions only 5 succeeded:
  • GFP_GOI
  • GFP_modul
  • GFP_TS
  • eGFP_TS
  • eGFP_module


Wednesday

Today we have run 8 reaction, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.

Only one succeeded:
  • hgH-polyA

August


Week 5: 01.08.2011 - 07.08.2011

Monday

New week, new energy! Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!

New biobricks in the bag:
  • RFP_GOI (20)
  • RFP_modul (21)
  • RFP_TS (22)
  • bleR (29)
  • pgpdA (26)
  • yA (27)
  • Plasmid_fun (28)
  • Plasmid_mam (30)
  • Amp cas  (C1)
  • Hygromycin (18)
  • CYP79A2 (Copenhagen part) (C4)
  • Terminator (Copenhagen part) (C6)
  • pIPTG (Copenhagen part) (C2)
  • CYP79B2(Copenhagen part) (C5

Wednesday

We still have 2 biobricks we haven’t been able to obtain with PCR: pyrG and pyrG-DR.
Today we tried again with gradient PCR to find the optimal melting temperature, but it did not help.

Thursday

Today we purified 22 biobricks, which have been obtained with PCR during the last week of laboratory work. The purification of DNA was done by using the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) according to the manufactures manual for purification of DNA from TAE and TBE agarose gels protocol, see protocol section for more information.

Despite several attempts, we have not been able to obtain biobrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, meaning that the DNA template was the source of error.

We also started cultivation of the mammalian cell line, U2OS, which is derived from a 15-year old patient with osteosarcoma (bone cancedr). They were defrosted and cultivated in in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.

Week 5: 08.08.2011 - 14.08.2011


Monday

Today we made the first USER cloning, it is very exciting to see if our system works!
All our biobricks are divided into devices, see protocol for USER MIX.

The following devices were cloned today:
  • Device 1: plasmid_mam (30) + CMV (3) + eGFP (32) + Hgh pA (33) + Hygromycin (18)
  • Device 2: plasmid_mam (30) + SV40 (2) + eGFP (32) + SV40 pA (4) + Hygromycin (18)
  • Device 3: plasmid_mam (30) + PGK (1) + eGFP (32) + BGH pA (5) + Neomycin (19)
  • Device 4: plasmid_fun (28) + pAlc (7) + LacZ (8) + T1 (14) + argB (17)
  • Device 5: plasmid_fun (28) + gpdA (26) + GFP (24) + trpC (13) + hygR (12)
  • Device 6: plasmid_fun (28) + gpdA (26) + RFP (21) + trpC (13) + hygR (12)
  • Device 7: plasmid_mam (30) + pIPTG (C2) + CYP79B2 (C5) + Terminator (C6) + amp (C1)
  • Device 8: plasmid_mam (30) + pIPTG (C2) + CYP79A2 (C4) + Terminator (C6) + amp (C1)

PCR reactions done today:
  • eGFP+k_GOI (6)
  • eGFP_TS (31)
  • yA (27)
  • GFP_TS (25)
  • RFP_TS (22)
  • RFP_GOI (20)
  • MTS (34)
  • GFP_PTS1_fun (35)
  • GFP_PTS1_mam (36)
  • RFP_NLS_module (37)
  • pyrG (10)
  • pyrG-DR (11)


After new DNA template we finally succeed in getting biobricks pyrG and pyrG-DR.
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.

Tuesday

We inoculated E.coli with the plasmides done by USER cloning yesterday.

PCR done today:
  • RFP_TS
  • RFP_GOI
  • RFP_NLS_Module
  • nr.37



Purification with GFX kit on the following biobricks:
  • nr.6
  • nr. 31
  • nr.27
  • nr.25
  • nr.22
  • nr.34
  • nr.35
  • nr.36
  • nr.20
  • nr.37
  • nr.10
  • nr.11

USER cloning done today:
  • Device 9: plasmid_fun (28) + xxx (26) + xxx (34) + xxx (25) + xxx (13) + xxx (12)
  • Device 10: plasmid_fun (28) + xxx (26) + xxx (35) + xxx (13) + xxx (12)
  • Device 11: plasmid_fun (28) + xxx (26) + xxx (37) + xxx (13) + xxx (12)
  • Device 12: plasmid_mam (30) + xxx (3) + xxx (36) + xxx (5) + xxx (18)
  • Device 13: plasmid_mam (30) + xxx (3) + xxx (32) + xxx (32) + xxx (18)

Wednesday

The U2OS cells are checked out in the microscope. They had a confluency of about 90%, which means that they are ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")

Friday

Today we did 13 PCR reaction, some of them includes parts to characterise our two promoters pAlc and DMKP-P6 and some parts for iGEM Copenhagen team.

  • GFP_GOI (23)
  • GFP_PTS_fun (35)
  • GFP_PTS_mam (36)
  • RFP_NLS (37)
  • DMKP_P6 (38)
  • ptrA (40)
  • 1A2 (C7)
  • 2C9 (C8)
  • pAlc-L (B1)
  • DMKP_P6-L (B3)
  • RFP_TS (22)
  • RFP_GOI (20)




The U2OS cells are splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.

Week 7: 15.08.2011 - 21.08.2011


Monday

The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They are placed in the incubator overnight - and tomorrow they will be ready for transfection with plasmides.
Preparing for characterization of two promoters with β-galactosidase assay were the plasmid p68 cut with restriction enzyme.

Purification of PCR products done last Friday were done with GFX kit.

9 PCR reactions were done today
  • eYFP_module (41)
  • eYFP_GOI (42)
  • eYFP_TS (43)
  • eCFP_module (47)
  • eCFP_GOI (48)
  • eCFP_TS (49)
  • mCheryy_module (44)
  • mCherry_GOI (45)
  • mCHerry_TS (46)




USER cloning done today and transformed in E.coli today:
  • Device 21: plasmid_fun (28) + xxx (7) + xxx (8) + xxx (14) + xxx (40)
  • Device 22: plasmid_fun (28) + xxx (26) + xxx (24) + xxx (13) + xxx (10)
  • Device 23: plasmid_fun (28) + xxx (26) + xxx (35) + xxx (13) + xxx (10)
  • Device 24: plasmid_fun (28) + xxx (26) + xxx (21) + xxx (13) + xxx (10)
  • Device 25: plasmid_fun (28) + xxx (26) + xxx (37) + xxx (13) + xxx (10)
  • Device 26: plasmid_fun (28) + xxx (26) + xxx (34) + xxx (25) + xxx (13) + xxx (10)
  • Device 28: plasmid_fun (28) + xxx (38) + xxx (23) + xxx (22) + xxx (15) + xxx (29)
  • Device 29: plasmid_fun (28) + xxx (7) + xxx (20) + xxx (22) + xxx (16) + xxx (11)
  • Device 13: plasmid_mam (30) + xxx (3) + xxx (32) + xxx (5) + xxx (18)
  • Device 12: plasmid_mam (30) + xxx (3) + xxx (36) + xxx (5) + xxx (18)
  • Device 17: plasmid_mam (30) + xxx (1) + xxx (6) + xxx (31) + xxx (33) + xxx (19)

Tuesday

We transfected U2OS cells with plasmides containing GFP and placed them in the incubator.

USER cloning done today and transformed in E.coli today:
  • Device 14: plasmid_mam (30) + xxx (3) + xxx (41) + xxx (5) + xxx (18)
  • Device 15: plasmid_mam (30) + xxx (3) + xxx (44) + xxx (5) + xxx (18)
  • Device 16: plasmid_mam (30) + xxx (3) + xxx (47) + xxx (5) + xxx (18)
  • Device 18: plasmid_mam (30) + xxx (1) + xxx (42) + xxx (43) + xxx (33) + xxx (19)
  • Device 19: plasmid_mam (30) + xxx (2) + xxx (45) + xxx (46) + xxx (4) + xxx (19)
  • Device 20: plasmid_mam (30) + xxx (2 + xxx (48) + xxx (49) + xxx (4) + xxx (19))
  • Device 21: plasmid_fun (28) + xxx (7) + xxx (8) + xxx (14) + xxx (40)

Wednesday


Inoculations of E.coli transformants with USER devices done the day before.

Restriction analysis of Device 4, 7, 8 and 27

Insert of gel-photo 17.08.11 09:35

USER cloning done today and transformed in E.coli today:
  • Device 4: plasmid_fun (28) + xxx (7) + xxx (8) + xxx (14) + xxx (17)
  • Device 21: plasmid_fun (28) + xxx (7) + xxx (8) + xxx (14) + xxx (40)
  • Device 22: plasmid_fun (28) + xxx (26) + xxx (24) + xxx (13) + xxx (10)
  • Device 23: plasmid_fun (28) + xxx (26) + xxx (35) + xxx (13) + xxx (10)
  • Device 24: plasmid_fun (28) + xxx (26) + xxx (21) + xxx (13) + xxx (10)
  • Device 25: plasmid_fun (28) + xxx (26) + xxx (37) + xxx (13) + xxx (10)
  • Device 26: plasmid_fun (28) + xxx (26) + xxx (34) + xxx (25) + xxx (13) + xxx (10)
  • Device 28: plasmid_fun (28) + xxx (38) + xxx (23) + xxx (22) + xxx (15) + xxx (29)
  • Device 29: plasmid_fun (28) + xxx (7) + xxx (20) + xxx (22) + xxx (16) + xxx (11)
  • Device 31: plasmid_mam (30) + xxx (C2) + xxx (C7) + xxx (C6) + xxx (C1)
  • Device 32: plasmid_mam (30) + xxx (C2) + xxx (C8) + xxx (C6) + xxx (C1))
  • Device 7: plasmid_mam (30) + xxx (C2) + xxx (C5) + xxx (C6) + xxx (C1)
  • Device 8: plasmid_mam (30) + xxx (C2) + xxx (C4) + xxx (C6) + xxx (C1)


Purifications of plasmid from transformed E.coli with mini-prep and restriction analysis of the plasmids.

Thursday

The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.

Friday


USER cloning done today and transformed in E.coli today:
  • Device 28: plasmid_fun (28) + xxx (38) + xxx (23) + xxx (22) + xxx (15 + xxx (29)
  • Device 22: plasmid_fun (28) + xxx (26) + xxx (24) + xxx (13) + xxx (10)
  • Device 30: plasmid_fun (28) + xxx (7) + xxx (27) + xxx (22) + xxx (13) + xxx (C1)


    • Week 8: 22.08.2011 - 28.08.2011


    Monday

    The U2OS cells are splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.

    Tuesday

    The U2OS cells are transfected with all the mammalian plasmides. So hopefully we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.

    Measurements of plasmids with our device for sequencing. The measurement were done with Qubit Fluorometer from Invitrogen.

    And more restriction analysis of plasmids were done.

    Wednesday

    The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great - everything worked = we are done with the mammalian cells. Hip hip hurra!

    September

    Week 9: 29.08.2011 - 04.09.2011

    Tuesday

    All plasmids and primeres were sent to sequencing.

    Wednesday

    PCR reaction of the shipping plasmid were done. The shipping plasmid had to modified with our USER tails so we quickly could clone our 2 biobricks and one device to send.



    Thursday

    USER cloning of the two promoters and device 12 with the shipping plasmid were done.

    Due to the extensive use of biobrick plasmid_fun(28) and plasmid_mam(30) we did a PCR reaction again so it can be sent to iGEM.

    Week 10: 05.09.2011 - 11.09.2011

    Monday

    Restriction analysis of shipping plasmid, to check if the USER cloning were done correct. Shipping plasmid were sent to sequencing.

    Inoculation of fungi for β-galactosidase and Bradford assay Wednesday and Fluorescence microscope Tuesday.

    Tuesday

    Today we looked at our fungi in fluorescence microscope, and what a beautiful sight it was! We have now prooven our system works in fungi as well as mammalian cells.

    Wednesday

    Today we spent all day in the lab with β-galactosidase and Bradford assay for charactisation of our two promoters.

    Friday

    Hopefully this day will be one of the last in lab. We are measuring the DNA concentration of our biobricks so they can be sent to MIT next week.

    October

    November

Retrieved from "http://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes"