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

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
insert 20.07.2011 07:12

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
Insert gel-photo 21.07.2011 14:31

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, adding MgCl2 50mM to the reactions.

Biobrick obtained:
  • CMV
  • hygR
insert 22.07.11 12:11 insert 22.07.11 12:27

The fragments will be frozen and purified later.

Week 4: 25.07.2011 - 31.07.2011


Monday

Got a new batch of primeres today, they need to be diluted and stocked. There were still 5 biobricks we had difficulty in obtaining; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We tried to run a touch PCR program, where each sample will run with a annealing temperature from 55° to 65° in each cycle.

The PCR reactions succeed and we got the following biobricks:
  • SV40 +ori
  • SV40 pA
  • LacZ
Insert of gel photo 26.07.11 11:45 The fragments will be frozen and purified later. We still have trouble with obtaining pyrG and pyrG-DR.

Tuesday

Many new biobricks will be amplified today with the new batch of primeres we received yesterday.

Out of 15 PCR reactions only 5 succeeded:
  • GFP_GOI
  • GFP_modul
  • GFP_TS
  • eGFP_TS
  • eGFP_module
Insert gel photo 26.07.11 16:24

Wednesday

8 PCR reactions done today with both standard and touch PCR on the biobricks that we still have some problems obtaining.

Only one succeeded:
  • hgH-polyA
Insert 27/7-11

August


Week 5: 01.08.2011 - 07.08.2011

Monday

A new week, new energy! Today we did 15 PCR reactions were conducted including parts for iGEM Copenhagen team and we got 14 correct. The annealing temperature was change to 63° and it worked like magic!

New biobricks in the bag:
  • RFP_GOI
  • RFP_modul
  • RFP_TS
  • bleR
  • pgpdA
  • yA
  • Plasmid_fun
  • Plasmid_Mam
  • Amp
  • Hygromycin
  • CYP79A2 (Copenhagen part)
  • Terminator (Copenhagen part)
  • pIPTG (Copenhagen part)
  • CYP79B2(Copenhagen part)

Wednesday

We still have 3 biobricks we haven’t been able to obtain with PCR: Neomycin, pyrG and pyrG-DR.
Today we tried again with gradient PCR to find the optimal melting temperature. We were only one new biobrick:
  • Neomycin
Insert gel photo 03.08.11 16:24

Thursday

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

Still have not been able to get biobrick pyrG and pyrG-DR after many many attempts. So we did use some other primers without USER-tails (Which sometimes can be tricky using) and have been used before and are known to work. This was done to verify if there was something wrong with the DNA template. The gel electrophoresis did not show any bands, so we concluded that it is our DNA template that is the source of error with the PCR reaction.

Today, we also started cultivation of the two mammalian cell lines, HeLa and U2OS. They were defrosted and cultivated in in DMEM medium containing serum, penicillin and streptomycin. Each 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 started out first USER cloning, 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)
insert gel photo 08.08.11

After new DNA template we finally succeed in getting biobricks pyrG and pyrG-DR.
U2OS cells HeLa cells

Tuesday

Inoculation of E.coli with USER cloning done the day before.

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

Inser gelphoto 09.08.11 x2

Purification with GFX kit on rhe 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

U2OS cells HeLa 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)


inset gel-photo 12.08.11

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 are splitted and passed on to coverslips placed in a 6-well plate and a new 75 cm2 flask. They are placed in the incubator overnight - and tomorrow they will be ready for transfection.

Tuesday

Transfection of U2OS cells.

September

October

November

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