Laboratory Notebook

15-06-09

digest glgA/B plasmids (stored in #SZKD#)

15-06-10

• after digest glg A and B:
  • A and B will be ligated in pSB1K30 (ligation tubes: A2 and B2)
  • stored in #QHHR#
• transformation into DH5α

15-06-11

• master plates of glgA, glgB in pSB1K30 (LB+K)

15-06-12

• colony PCR on glgA/B constructs to identify positive clones
• make overnight cultures of glgA/B constructs

15-06-13

• make cryos of glgA/B constructs (table above)
• plasmid prep of glgA, glgB (table above)

15-06-15

• measured concentrations of purified glgA/glgB constructs
• heat shock of glgA (#D6VB#, #RLDN#) and glgB (#ATE6#) in pSB1K30 into BL21(DE3)

15-06-16

• BL21 transformation master plates (glgB transformation was not successful)

15-06-17

Digestions with 40' digest and 20' inactivation

(previous digests A2, B2, pSB1K30 were discarded from #SZKD# because agarose gels were really ugly)

Ligations and transformations

15-06-18

• make master plates and liquid cultures of transformations (glgA in pSB1K30, glgB in pSB1K30; 6 liquid, 12 plate)

15-06-19

Colony PCR on glgA,B,C constructs

Cryos

Master plates of glgA, glgB clones that were white under green light.

15-06-22

• prep good plasmids of glgA (table above)
• pipette glgA in pSB1K30 for sequencing (#A9W9# and #XE3D#)
• colony PCR on glgA, glgB plates from Saturday (#A9W9# and #XE3D#)

• overnight cultures of good clones from the glgB colony PCR (LB+K)

15-06-23

• cryos and plasmid prep from overnight cultures of glgB
• the plasmid prep went wrong and resulted in concentrations < 10 ng/µl

• overnight cultures of glgB in pSB1K30 from the master plate

15-06-24

• in the morning: freeze overnight culture of glgB
• in the afternoon: plasmid prep of glgB (re-used the existing ID from the table of Monday)
• pipette glgB for sequencing

15-06-26

Digestions

15-06-29

Ligation and Transformation (first try)

>> #LVVK#:4 (pSB1K30) looked bad on agarose gel! Do the ligation again with pSB1K30 from a different tube!

Ligation (second try, only 1 µl of K30)

• transformation is planned for the day after

15-06-30

• heat shock of K1585312 in pSB1K30 ligation product (5 µl #RRCV#:2) into 40 µl BL21 Gold (DE3)

15-07-01

• master plate of K1585312 in pSB1K30 (BL21 Gold DE3)

15-07-02

• colony PCR of K1585312 in pSB1K30 (desired: 1683, negative: 1227)
• overnights of good clones

colony PCR with KAPA 2G

conditions for 30 cycles:

Digestions

Ligation

15-07-03

• cryo cultures
• plasmid prep
• pipette for sequencing

15-07-06

• transformation of #RRCV#:2B into BL21 Gold (DE3)
• decided to use pSB1A30 because the difference between induced and non-induced pSB1K30 could not be seen on SDS gels

PCR 1 to subclone glgC into pSB1A30: backbone amplification

PfuS PCR

conditions for 30 cycles:

15-07-08

• master plate (LB+K+IPTG) of glgC in pSB1K30 transformations

PCR2 to subclone glgC into pSB1A30: glgC amplification

PfuS PCR

conditions for 30 cycles:

15-07-09

PCR product purification

CPEC assembly of glgC in pSB1A30

5 µl #SWMB#:II were heat shocked into BL21 Gold (DE3) and plated on LB+A

15-07-10

• make masterplates (LB+A) and overnights of glgC in pSB1A30

15-07-11

• cryo cultures
• freeze culture

• all clones except #6 were red

15-07-13

• plasmid prep of glgC in pSB1A30 clone #6 in #VMN3#
• new overnights of white clones on masterplate: clone #9 and #10

15-07-14

• plasmid prep of glgC in pSB1A30 clone #9+10 (table above)
• all three glgC in pSB1A30 plasmids went into sequencing

15-07-15

• analysis of sequencing results
• 2 precultures of glgC in pSB1A30 from clone #6, one of RFP in pSB1A30 #6DA3#

Expression

15-06-30

glgA Plated #W8VB# and #KK16# on LB+K

15-07-01

• make overnight precultures from the plate (2x #W8VB#, 1x #KK16# -> A,B,C)
• reserve 3x 500 ml shake flasks

15-07-02

08:50 inoculate 50 ml main cultures LB+K (labelled A,B,C) with 1 ml of culture

monitor the OD and induce cultures A+B with IPTG at OD=0.6

5 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

10 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

24 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

OD monitoring (numbers show time in hours)

15-07-03

• take the final sample
• prepare SDS-PAGE (cooked samples in #B13F#)
• run SDS-PAGE

15-07-06

glgB Plated #Q6Z1# on LB+K

15-07-07

• make overnight precultures from the plate (2x Q6Z1, (1x KK16) -> A,B,C)
• reserve 3x 500 ml shake flasks

15-07-08

08:55 inoculate 50 ml main cultures LB+K (labelled A,B,C) with preculture to an OD of 0.1

monitor the OD and induce cultures A+B with IPTG at OD=0.6

5 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

10 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

24 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

OD monitoring (numbers show time in hours)

15-07-09

• cooked SDS samples in #6SOX#
• run SDS page

15-07-15

• glgC overnight of clone # from Masterplate of glgC in pSB1A30

15-07-16

09:15 inoculate 25 ml main cultures LB+A (labelled A,B,C) with preculture to an OD of 0.1

monitor the OD and induce cultures A+B with IPTG at OD=0.6

5 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

10 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

24 h after induction from each culture take 2x 2 ml sample (centrifuge, discard supernatant and freeze)

OD monitoring (numbers show time in hours)

15-07-17

• cooked SDS samples of glgC expressio are in #FWCT#
• SDS PAGE of glgC expression tests

SDS PAGE of glgC expression glgC was expressed in pSB1A30 and compared to mRFP expression. The small arrows point to the GlgC bands of 48 kDa. No difference between induced and uninduced was observed. After overnight expression the strong bands were no longer present.

References