Team:Potsdam/Notebook

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Lab book

We separated our lab book into the following parts:




Wednesday, 07/12/17

*Preparation of Media:

Tryptone 35g
Yeast extract 17,5g
NaCl 17,5g
dd water 3,5L
*1.5 L were aliquoted: 2x 500 mL, 5x 100 mL
* 2 L were used for LB-Agar:

(notes: 500 mL LB-Agar are stored in 1L bottles --> this is necessary, because the LB-agar has to be heated in the microwave before preparing LB-agar plates)


Tryptone 16g
Yeast extract 10g
NaCl 5g
dd water 1L


-->aliquoted 2x500 mL (will be used for the preparation of competent cells)

Plating of JM109 ("Preplate" for "Preculture" which is needed for the preparation of competent cells)

Organization of boxes for Plasmids, Primer, Stocks, etc. --> the boxes are organized according our 3 main tasks: LLPS, high copy, low copy + antibiotics

Thursday, 07/13/17

-tried SLiCE buffer, failed miserably
-finished Triton buffer, inoculated culture for competent cells
-prepared DTT (1M at -20C), forgor to do aliquots
-finished competent cell buffer

Friday, 07/14/17

-aliquoted DTT stock in 5x0,9ml
-sterile filtrated CaCl2 solution
-sterile filtrated CaCl2 + Glycerol
-made competent cells following protocol
- made 23 LBagar plates with CAP
-made 31 agar plates with AMP and 23 with KAN


Tuesday, 07/18/17

-started competent cell test by transferring test kit DNA (BBa_J04450) into our competent cells using Competent Test Kit protocol inoculated SOC medium (from Fabian) with JM109 -prepared 10x SLiCE buffer (19 50 µl aliquots in the -20° freezer)

Wednesday, 07/19/17

-Start with the JM109 culture for preparation of the SLiCE extract (bacteria had to grow until they reach an OD600nm of 3.0 -->reached at ca. 12:00i)
-Preparation of 80 % glycerol solution (sterile filtered)
-Preparation of 100 mM CaCl2 solution (autoclaved)
-Plan for the next days
-Further preparation of the SLiCE extract

Thursday, 07/20/17

-inoculated JM109 preculture for competent cells tomorrow with 20 mL LB
-prepared eppis for competent cells tomorrow
-competent cell test following competent cell test protocol
-filled 1mL tip boxes, glass beads for plating, sent for autoclaving

Friday, 07/21/17

-made competent cells
-tested competente cells on AMP plates
              a) pUC19 10pg/ul = 0,01 ng/ul
              testet competente cells with 1 ul from a)

Tuesday, 07/25/17

-cfu calculated

Count if colonies
1*attenuated 10*attenuated 100*attenuated
Sample A
 796 112 4
Sample B
 928 112 17
1*attenuated 10*attenuated 100*attenuated
Sample A
 7,96*10^9 colonies/ng
 1,12*10^10 colonies/ng
 4*10^9 colonies/ng
Sample B
 9,28*10^9 colonies/ng
 1,12*10^10 colonies/ng
 1,7*10^10 colonies/ng


Thursday, 08/10/17

-made 1L 10x TBE buffer:

T1M Tris-HCl 121,14g
1M Boric acid 61,82g
0,02M EDTH 7,44g


-restock with ddwater to 1L
-pH: 8,5

Tuesday, 08/15/17

*made new 10xTBE buffer (1L)
*same as above but used Tris Base

Thursday, 08/17/17

-prepared plates with Kan, Cap resistance
-added the antibiotica right before pouring the plates under clean bench for 500 ml LB -Agar + ad 500 uL antibiotica
-> stored in the freezer room under the table Cap plates and on the table Kan plates

Thursday, 09/07/17

-prepared medium for plates

Friday, 09/08/17

-prepared 50ml 0,02 M NaOH
-prepared plates with KAN

Thursday, 09/28/17

-0,7% Agarose
(used: 500ml 1xTBE, 3,5g Agarose, 5µl EtBr)

Wednesday, 10/04/17

-ampicillin-LB-plates

Tuesday, 10/17/17

-media for LLPS (YPDA+Ura+Tryp (2*750ml)