Laboratory Notebook
15-05-18
Amplification of gBlocks by Phusion PCR:
| step |
temperature [°C] |
duration
|
| initial denaturation |
98 |
0'30"
|
| denaturation |
98 |
0'30"
|
| gradient annealing |
55-67 |
0'30"
|
| elongation |
72 |
0'46"
|
| final elongation |
72 |
5'00"
|
3x 20 µl reactions, 20 ng template DNA
| template |
Primer_F |
Primer_R |
Annealing Temperature [°C] |
Product length [bp] |
Elongation time |
Result
|
| mdh gBlock #MDHG# |
#B8WH# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
1259 |
0'38" |
No bands at all
|
| hps gBlock #8D18# |
#B8WH# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
656 |
0'20" |
...
|
| phi gBlock #HPSG# |
#B8WH# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
737 |
0'22" |
...
|
| xpk 1 gBlock #XPK1# |
#B8WH# |
#WNCS# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
1193 |
0'36" |
...
|
| xpk 2 gBlock #XPK2# |
#4SBT# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
1426 |
0'43" |
...
|
There was not enough Polymerase for all of the mastermixes, so Phi, Hps, Xpk1 and Xpk2 are in the freezer (falcon #CBPQ#)
- Gel electrophoresis (2log ladder) with Mdh amplification products, PCR tube with Mdh2 is in falcon #8K9Q#
No PCR product on gel for Mdh2 - The amplification of Mdh2 will be repeated with the other gBlocks tomorrow, when there's enough Polymerase again
15-05-19
Repeated Amplification of gBlocks by Phusion PCR:
| step |
temperature [°C] |
duration
|
| initial denaturation |
98 |
0'30"
|
| denaturation |
98 |
0'30"
|
| gradient annealing |
55-67 |
0'30"
|
| elongation |
72 |
0'46"
|
| final elongation |
72 |
5'00"
|
4x 50 µl reactions, 30 ng template DNA
| template |
Primer_F |
Primer_R |
Annealing Temperature [°C] |
Product length [bp] |
Elongation time |
Result
|
| mdh2 gBlock #MDHG# |
#B8WH# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
1259 |
0'38" |
2 weak bands
|
| phi gBlock #8D18# |
#B8WH# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
656 |
0'20" |
3 strong bands
|
| hps gBlock #HPSG# |
#B8WH# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
737 |
0'22" |
3 strong bands
|
| xpk 1 gBlock #XPK1# |
#B8WH# |
#WNCS# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
1193 |
0'36" |
3 strong bands, 1 weak band
|
| xpk 2 gBlock #XPK2# |
#4SBT# |
#3N6Z# |
55.1 (2) + 57.0 (4) + 59.9 (6) |
1426 |
0'43" |
3 strong bands (slight smear)
|
- check with gel electrophoresis
- PCR products stored in falcon #FOL9#
15-05-20
PCR Purification
| fragment |
from |
tubes |
purified plasmid |
concentration [ng/µl]
|
| mdh |
#FOL9# |
(C1, C2) |
#Z4QF# |
34.2
|
| phi |
#FOL9# |
D1, D2, D3 |
#NTXL# |
55.2
|
| hps |
#FOL9# |
E1, E2, E3 |
#B8ST# |
60.6
|
| xpk 1 |
#FOL9# |
F1, F2, F3 |
#6EPP# |
40.0
|
| xpk 2 |
#FOL9# |
G1, G2, G3 |
#99DW# |
38.8
|
- decide on how to proceed with Mdh2: PROCEED
- plasmid purification of J04450 in pSB1C3: #CD8B#; start restriction cloning for Mdh, Phi and Hps
- Repeat amplification of pSB1C3 with #TSTV#, #FYTO# (product: linear pSB1C3, #6WCN# - done by iSL, iLM and iMO)
Q5-CPEC with Xpk
#6EPP# and #99DW# into #6WCN#
| component |
volume [µl]
|
| water |
4,6
|
| Q5 buffer |
5
|
| DMSO |
0.75
|
| dNTPs (2 mM) |
5
|
| Q5 polymerase |
0.5
|
| backbone fragment (#6WCN#) |
0.8
|
| amplified xpk part I gBlock #6EPP# |
1,4
|
| amplified xpk part II gBlock #99DW# |
1,8
|
| elongation time |
2'17
|
- 98:0'30", 15x(98:0'10", 55:0'30", 72:2'20"), 72:10'00"
Restrictions
PCR-amplified and purified Hps (#B8ST#), Phi (#NTXL#) and Mdh2 (#Z4QF#) were digested with EcoRI and PstI -> products stored in #CQ1V#
Ligations
The backbone is pSB1C3 from digested #CD8B# in all cases. 2x the concentration of T4 Ligase due to human error.
Products stored in #4A4P#.
Transformations
- 5 ul of ligation or CPEC products by heatshock transformation
- transformation in PCR tubes:
- 1 = xpk, via CPEC (50 µl commercial NEB5a)
- 4 = phi, via ligation (100 µl NEB10b)
- 5 = hps, via ligation (50 µl NEB10b)
- 6 = mdh, via ligation (100 µl NEB10b)
- plated on LB+C
15-05-21
- plates with trafos from yesterday (15-05-20) look good
- order sequencing primers
- make master plates and overnights of mdh2, hps, phi and xpk clones (LB+C)
- each construct: 6 overnight cultures and 8 to 10 colonies on master plates
15-05-22
- cryos and plasmid purifications
| construct |
number |
cryo-IDs |
purified plasmid
|
| K1585210 (mdh) |
#1 |
#3WMA# |
#PXZS#
|
| |
#4 |
#YY8F# |
#LXD8#
|
| K1585211 (hps) |
#1 |
#HWXN# |
#ZTFH#
|
| |
#2 |
#Q8WZ# |
#PD3D#
|
| K1585212 (phi) |
#1 |
#CLE1# |
#CK4X#
|
| |
#2 |
#QD6Z# |
#AB8X#
|
| K1585213 (xpk) |
#1 |
#H9PV# |
#MWBY#
|
| |
#2 |
#6KC6# |
#3CW8#
|
| |
#3 |
#R8Y6# |
#64FM#
|
| |
#4 |
#BSAN# |
#WSBD#
|
- Pipet sequencing for every purified plasmid with Primers #A9W9# and #XE3D#
15-05-27
- Result of Sequencing: Nothing works
- Colony PCR of clones that were not sequenced, check the length of the insert. Primers #A9W9# and #XE3D# are used for every PCR.
- elongation time: 2:54 for xpk, 1:30 for phi, hps and mdh
- annealing T: 58 °C
| construct |
number on masterplate |
expected length [bp] |
valid?
|
| K1585210 (mdh) |
#6 |
1494 |
no
|
| |
#7 |
|
no
|
| |
#9 |
|
no
|
| |
#10 |
|
no
|
| K1585211 (hps) |
#3 |
972 |
no
|
| |
#6 |
|
no
|
| |
#7 |
|
no
|
| |
#8 |
|
no
|
| |
#9 |
|
yes
|
| |
#10 |
|
no
|
| K1585212 (phi) |
#3 |
891 |
yes
|
| |
#4 |
|
no
|
| |
#5 |
|
no
|
| |
#6 |
|
no
|
| |
#7 |
|
no
|
| |
#8 |
|
yes
|
| |
#9 |
|
yes
|
| |
#10 |
|
yes
|
| K1585213 (xpk) |
#5 |
2814 |
no
|
| |
#6 |
|
no
|
| |
#7 |
|
no
|
| |
#8 |
|
no
|
| |
#9 |
|
yes
|
The agarose gel pictures of this colony PCR can be found at Scieobo/WetLab/Documentation/Methanol.
15-05-28
MDH
- PCR amplification of mdh from gBlock -> product #64LF#
| component |
volume [µl]
|
| water |
202.5
|
| buffer B2 |
25
|
| dNTPs O2 |
5
|
| Primer #B8WH#:P':I7 |
5
|
| Primer #3N6Z#:P':I3 |
5
|
| Template #MDHG#:Pink:B3 |
5
|
| PfuS E2 |
2.5
|
elongation time: 40s
product length: 1259 bp
annealing T: 55.0 - 57.5 °C
cycles: 30
- gel control => all lanes have a strong band at 700 bp and a weak band at the correct length (1259 bp)
PHI
- plasmid purification from overnight cultures of clones #3, #8, #9 and #10
| clone # |
purified plasmid
|
| #3 |
#ANNA#
|
| #8 |
#HASI#
|
| #9 |
#ENKK#
|
| #10 |
#B3ZP#
|
HPS
- purify plasmid from overnight culture of clone #9, #H4M9#
- new Ligation: pSB1C3 from #CQ1V#:V with HPS digestion from #CQ1V#:III (if sequencing of #H4M9# fails)
- Ligation product in Falcon #SEXY# and ready for transformation
XPK
- purified plasmid from overnight culture of clone #2 and #9
| clone |
purified plasmid
|
| xpk in pSB1C3 #2 |
#DOOR#
|
| xpk in pSB1C3 #9 |
#BAEM#
|
- more xpk in pSB1C3 will follow => CPEC product in #4A4P# will be transformed again.
15-05-29
- Colony PCR of yesterday's xpk masterplates with Primers #A9W9# and #XE3D#
| Biobrick |
expected length |
successful clones
|
| BBa_K1585213 (Xpk) |
2814 |
#14, #23
|
- new amplification of mdh from gBlock #MDHG#:
elongation time: 39s
annealing T: (1) 57.3 anf (2) 59.0
reaction volume per tube: 20 µl
| component |
Volume [µl]
|
| H2O |
7.4
|
| 2x MM Q5 |
10
|
| Primer #B8WH# |
0.8
|
| Primer #3N6Z# |
0.8
|
| Template #MDHG# |
1
|
PCR products stored in Falcon #NEIN#
- gel control: again very ugly result, seems like amplification of mdh is not possible (gBlock defective?)
- falcon #NEIN# gets discarded
- master plates of hps ligation clones
15-05-30
| gene |
clone number |
cryo-ID |
plasmid-ID
|
| Xpk |
14 |
#T33B# |
#OFLR#
|
| Xpk |
23 |
#PLAE# |
#AMSX#
|
- sequencing of xpk plasmids
- colony PCR of hps (expected length: 972 bp):
| clone ID on 15-05-29 HPS masterplate |
band on colony PCR gel
|
| 1 |
none
|
| 2 |
none
|
| 3 |
none
|
| 4 |
300
|
| 5 |
none
|
| 6 |
none
|
| 11 |
none
|
| 12 |
300
|
| 13 |
none
|
| 14 |
700
|
| 15 |
300
|
| 16 |
300
|
According to colony PCR result, none of the clones could be validated
- masterplates of further white hps clones from trafoplates (20.5. and 28.5).
15-06-01
colony PCR of hps Clones
- gel control of Colony PCR
- successful clone #10, #42, #4
summary:
MDH: Mail to Brian Caudill about the problems with the gBlock was sent (not even amplification works)
XPK: Waiting for the sequencing results of four colony PCR validated clones.
HPS: Waiting for the sequencing results of one colony PCR validated clone. Colony PCR of further clones is running --> more clones that could be sequenced
PHI: Waiting for the sequencing results of four colony PCR validated clones.
15-06-02
plasmid purification of PCR validated HPS clones:
| Clone |
cryo |
plasmid |
plasmid concentration
|
| #4 |
#RFS3# |
#PEVS# |
123,6 ng/ul
|
| #10 |
#CXEX# |
#BZKF# |
112,0 ng/ul
|
| #42 |
#Y9QC# |
#ZVEM# |
145,0 ng/ul
|
15-06-03
hps: mutation free clone #9, cryo: #863A# and plasmid: #H4M9#
- discard: #CXEX#, #HWXN#, #N8DP#, #PB1E#, #Q8WZ#, #RFS3#, #Y9QC# done
xpk: no mutation free plasmids with Xpk, so new transformation of Xpk CPEC products in DH5alpha cells
phi: mutation free clone #3, cryo: #4DMS# and plasmid: #ANNA# and mutation free clone #9, cryo: #AE8V# and plasmid: #ENKK#
- discard: #CDD9#, #CLE1#, #FQYH#, #NLQL#, #QD6Z#, #RD8C#, #XADS#, #AB8X#, #CK4X#, #HASI#, #B3ZP# done
15-06-04
- transformation of xpk was not very succesful, just 4 colonies (even though do Masterplate and overnights)
- do CPEC again tomorrow
15-06-05
- colony PCR of the four transformants of xpk from 15-06-04 + gel picture: none has expected lenght
CPEC of #XPK1# and #XPK2# in #6WCN#
- do CPEC not with PCR amplified gBlocks but with pure gBlocks, reduces risk of mutations in the long coding sequence
cycler programme:
| step ! time |
temp [°C]
|
| I |
0'30 |
98
|
| II |
0'10 |
98
|
| III |
0'30 |
55
|
| IV |
2'17 |
72
|
| v |
10'00 |
72
|
| VI |
forever |
8
|
repeat step II to IV 15 times
CPEC pipetting
| component |
amount
|
| water [µl] |
0,3
|
| Q5 buffer [µl] |
5
|
| DMSO [µl] |
0,75
|
| dNTPs [µl] |
5
|
| Q5 Polymerase |
0,5
|
| pSB1C3 backbone (#6WCM#) |
0,8
|
| #XPK1# |
estimated 4,9 ul
|
| #XPK2# |
estimated 5 µl
|
| length [bp] |
4569
|
- #XPK1# and #XPK2# are empty now, last try or we have to order new gBlocks
- Products in falcon #AT19#
15-06-07
- transform CPEC product of xpk via heat shock transformation in DH5alpha
15-06-08
- make master plates of all clones
15-06-09
- colony PCR of first 72 xpk clones on masterplates; FW Primer #A9W9#; RV Primer #XE3D#
- expected length: 2815 pb
- Mastermix calculation for 80 samples:
| component |
amount
|
| water [µl] |
256
|
| #A9W9# (10µM) [µl] |
32
|
| #XE3D# (10µM) [µl] |
32
|
| one taq 2xMM [µl] |
400
|
successful clones: 1, 2, 4, 5, 6, 7, 14, 15, 16, 17, 19, 22, 23, 27, 29, 30, 31, 35, 37, 40, 41, 45, 46, 47, 48, 49, 54, 55, 58, 62, 68
- overnights and cryos of all successful clones
15-06-10
XPK plasmids
- make cryos, purify plasmids and send in for sequencing with two primers
| clone number |
cryo ID |
Plasmid ID |
Plasmid concentration
|
| #30 |
#AEWE# |
#ONNC# |
203.6
|
| #23 |
#TKD8# |
#HMHQ# |
302.5
|
| #7 |
#DQWO# |
#1BYO# |
343.2
|
| #27 |
#TFPT# |
#SP8K# |
252
|
| #16 |
#8WY9# |
#KFEY# |
306.5
|
| #45 |
#FZ9L# |
#B3B9# |
234.2
|
| #37 |
#Q4D3# |
#CTTL# |
353.5
|
| #35 |
#SZHH# |
#P1ER# |
164.8
|
| #22 |
#ERBM# |
#C3A3# |
303.9
|
| #46 |
#9ORT# |
#183P# |
335
|
| #40 |
#ARE4# |
#NF6D# |
334.6
|
| #47 |
#YX6L# |
#3BTM# |
297
|
| #15 |
#1MKB# |
#CRX1# |
343.3
|
| #54 |
#9AEZ# |
#4NMY# |
302.7
|
| #41 |
#SAP6# |
#6NLQ# |
349
|
mdh gBlock
- has the new mdh gBlock arrived yet? yes it has!
- 1000 ng of mdh gBlock were shipped
- diluted in 100 µl H2O; stored in #NFOE#
new restrictions:
- pSB1C3 backbone from #CD8B# and mdh gBlock from #NFOE# both digested with EcoRI and PstI
| Sample |
H2O [µl] |
10x NEBuffer [µl] |
EcoRI [µl] |
PstI [µl] |
DNA [µl]
|
| B (Backbone) |
14.4 |
2 |
0.4 |
0.4 |
2.8
|
| M (mdh gBlock) |
none |
2 |
0.4 |
0.4 |
20.0 (by mistake, 17.2 would have been correct)
|
- restriction products are stored in Falcon #PUFF#
Ligation
| component |
Volume [µl]
|
| Water |
11
|
| 10x T4 Ligase Buffer |
2
|
| Backbone Digest (B) |
2
|
| mdh digest (M) |
4
|
| T4 Ligase |
1
|
- product is stored in Falcon #PUFF#
- heat shock transformation into DH5alpha cells
- plate on LB
15-06-11
MDH
No clones on trafo plate!!
New transformations
- electroporation and heatshock
- plate both samples on agar plate LB+C
1. Electroporation:
- 25 µl electrocompetent cells
- 3 µl of Ligation product (#PUFF#:Lig)
- 800 µl SOC media
- 1.5 h regenerative incubation at 37 °C
2. Heatshock:
- 50 µl NEB10beta
- 5 µl of Ligation product (#PUFF#:Lig)
- 200 µl SOC media
- incubation for 1h at 37 °C
- plating and incubation at 37 °C over night
15-06-12
MDH
- make masterplates and overnights of new mdh transformation plates
XPK
Are the sequencing results ready? And if yes, how do we have to proceed with xpk?
- sequencings of #KFEY#, #B3B9#, #P1ER#, #C3A3#, #NF6D#, #CRX1# and #4NMY# were O.K. (but not with all necessary primers to check whole sequence)
- full sequencing results for these clones will be present at wednesday next week (15-06-17)
15-06-13
MDH
Mastermix calculation:
| component |
Volume [µl]
|
| ddH2O |
60.8
|
| 2x one taq MM |
95
|
| #A9W9# |
7.6
|
| #XE3D# |
7.6
|
PCR program:
| step |
temperature [°C] |
duration
|
| initial denaturation |
94 |
3'00
|
| denaturation |
94 |
0'15
|
| annealing |
58 |
0'15
|
| elongation |
68 |
1'30
|
| final elongation |
68 |
2'00
|
30 cycles
- gel control: bands at expected lenght clone #1, #4, #5, #14
- make cryos of colony PCR validated clones
- prepare colony PCR validated clones for plasmid purification
- update validity
15-06-15
MDH
- Can we trust the gel picture? Or should we do a CPEC to clone the mdh gBlock into pSB1C3 again? (linear pSB1C3 in #6WCN#)
Jonas:"I think we should do a CPEC today, just to be sure to have a backup to proceed with on wednesday. If sequencing does not give valid results on wednesday, we can do a colony PCR of the new clones and purify plasmid of the colony PCR validated clones."
Plasmid Purification
- plasmids of Clone #1 (#AARV#), #4 (#VRO8#), #5 (#9PBD#), #14 (#YSVQ#)
Sequencing of mdh
- full sequencing of the clones of mdh, each with A9W9, XE3D, MSP2
| clone number |
cryo ID |
validity |
plasmid ID |
plasmid concentration [ng/µl]
|
| #1 |
#DYQX# |
(yes) |
#AARV# |
??
|
| #2 |
#ASXH# |
no |
discard cryo and plasmid |
-
|
| #3 |
#PVMX# |
no |
discard cryo and plasmid |
-
|
| #4 |
#PR9O# |
(yes) |
#VRO8# |
??
|
| #5 |
#WNFY# |
(yes) |
#9PBD# |
??
|
| #6 |
#M4Y9# |
no |
discard cryo and plasmid |
-
|
| #7 |
#PLPD# |
no |
discard cryo and plasmid |
-
|
| #8 |
#MQMC# |
no |
discard cryo and plasmid |
-
|
| #9 |
#WM9K# |
no |
discard cryo and plasmid |
-
|
| #10 |
#8CFQ# |
no |
discard cryo and plasmid |
-
|
| #11 |
#XR8N# |
no |
discard cryo and plasmid |
-
|
| #12 |
#XWX8# |
no |
discard cryo and plasmid |
-
|
| #13 |
#PTOL# |
no |
discard cryo and plasmid |
-
|
| #14 |
#E3KY# |
(yes) |
#YSVQ# |
??
|
| #15 |
#1RW6# |
no |
discard cryo and plasmid |
-
|
- CPEC for mdh gBlock #NFOE# and linear pSB1C3 backbone #6WCN#
- do the CPEC and transformation on monday
- pick colonies make masterplate and overnights on tuesday
- do colony PCR on wednesday and purify the plasmid of validated clones if the sequencing of the 4 clones is negative
| component |
volume [µl]
|
| water |
6.9
|
| Q5 buffer |
5
|
| DMSO |
0.75
|
| dNTPs (2mM stock) |
5
|
| Q5 Polymerase (2U/µl) |
0.5
|
| #6WCN# |
0.8
|
| #NFOE# |
6.1
|
| step |
time |
temperature [°C]
|
| denature |
0'30 |
98
|
| denature |
0'10 |
98
|
| anneal |
0'30 |
55
|
| elongate |
1'40 |
72
|
| elongate |
10'00 |
72
|
| store |
forever |
8
|
- CPEC product stored in falcon #JAJA#
- 15 cycles
- transformation via heatshock, plate on LB+C
15-06-16
- make masterplates and overnight cultures of new mdh transformants
- we have 30 overnights, and all in all over 60 clones on masterplates. plasmids from overnights can be purifed tomorrow if sequencing is negative
15-06-17
- check sequencings of mdh and xpk
- remove unnecessary plasmids and cryos from the freezers
Both mdh and xpk had successful and mutation free clones; Part I. Biobricks is finished
References