Team:UCL/Labbook/Week16

Bacterial Labs

Monday 16th September

Results of the inoculations of transformations of 13/09 in 4xcmp: all showed growth apart from falcons 5.3, 5.2, 1.14 and 1.2. Glycerol stocks of the rest of 19 inoculations were made.

Minipreps of the above inoculations were made only for 1.3, 1.7, 1.9, 1.10, 1.11, 1.15, 1.20 and 5.1 due to lack of chromatographic columns.

Nanodrop result of the above minipreps showed concentration values below 16.0 ng/ul. Analytical digest of miniprep samples 1-9 (prepared the day before) as well as of 5.1 and 1.15 (which showed concentrations of about 15 ng/ul) with E and P

Components	Volume (ul)
DNA	5
EcoR1	1
Pst1	1
Buffer 3	1
BSA	0.5
dH2O	1.5
Total	10

Prep digest of miniprep of J63009 with E & P in order to keep up the pSB1C3 stocks

Components	Cut	Uncut	Control - pSecTag2A
J63009DNA	25	5	10
EocR1	2	0	0
Pst1	2	0	0
Buffer 3	4	0	0
BSA	0.5	0	0
dH2O	6.5	5	0
Total	40	10	10

These were incubated for 2 hours at 37O C. Casey ran a gel of the following samples in the next order: Zeo BB: 26D, 26u; 28D, 28u; AuxD, AuxU

Gel1: E+P double digest and P, single digest (Auxin bb): 3, 12, 13, 15. Gel2: D digest 26, 28 and Auxin bb. Minipreps: 26, 28, 3, 12, 13, 15, 21 (sample of which DNA is waiting to be eluted)

Tuesday 17th September

Miniprep of the 16 tubes of inoculations of MMP9 as well as tube 21 of which DNA had to be eluted.

Nanodrop results of the minipreps

Tube no.	ng/ul	260/280
1	20.6	1.9
2	26.9	1.64
4	22.0	3.2
5	4.8	2.16
6	22.7	2.99
7	16.4	2.41
8	16.9	2.01
9	7.1	1.49
10	27.3	1.79
11	16.0	1.72
12	22.2	1.41
13	11.0	1.73
14	12.0	1.6
15	18.4	1.57
17	45.1	1.62
18	9.2	1.72
21 (kc)	21.3	1.81

Nanodrop readings (Tom's)

Sample	ng/ul	260/280
J63A (glyc stock)	62.3	1.74
CCB4 (4xcmp comp cells)	46.8	1.94
CCB2 (2xcmp comp cells)	57.6	1.87

Analytical digest of zeo+pSB1C3 (from AA1 ligation) potential clones with xba1 Eight clones (AA1 col x miniprep 15/09 RC) + AA1 5xcmp mini prep were digested with xba1 following the recipe:

Components	Volume (ul)
DNA	5
Xba1	1
Buffer 2	1
BSA	0.5
dH2O	2.5
Total	10

Samples were briefly centrifuged and incubated at 37O C for circa 3 hours. After that, samples were supplemented with 3 ul dye and run on a gel.

Purification of 8 PCR reactions to amplify Zeo and BB hangers

These reactions (total volume of 400 ul) were left in the thermocycler at 4O C overnight. The samples were run on a gel together with 70 ul dye and the correct bands (1.8 kb) were gel extracted. This gel was purified and eluted in 40 ul Elution Buffer. The nanodrop readings of these were of 91.9 ul/ul with purity (260/280) of 1.96.

Preparative digest of amplified zeocin using EcoR1 and Pst1

Components	Volume (ul)
DNA	35
EcoR1	7
Pst1	7
Buffer 2	10
BSA	4
dH2O	37
Total	100

These reactions were Incubated for 2 hours at 37OC. After, the digest was purified with PCR purification kit. Nanodrop result was found to be 60.4 ng/ul (260/280=1.82, the purity).This was taken further for a ligation as per the following recipe:

Ligation 5 for zeocin and pSB1C3

pSB1C3 concentration = 50 ng/ul

Zeocin insert concentration = 25 ng/ul

Component	Lig 1 (ul)	Lig 2 (ul)	Lig 3 (ul) control	Lig 4 (ul) control
pSB1C3	2	2	2	0
Zeo	2	2.5	0	2
Quick T4 ligase	1	1	1	1
T4 ligase buffer	10	10	10	10
dH2O	5	4.5	7	7
Total	20	20	20	20

These tubes were incubated at room temperature for 5 minutes then used 5 ul from each for transformation using W3110 cells. Plated 10 ul, 90 ul on 5xcmp selective plates and then incubated overnight at 37°C. Next day, there was no growth on neither plates

Two gel were loaded using xba1 digests of recombinant zeo candidates

Ten ul of sample 1(showed correct band pattern) was used from glycerol stock to make an inoculation in 4xcmp 10 ml LB broth.

Inoculation of MMP9 glycerol stocks

These were made using 2 ml LB broth, 8 ul material from the glycerol stock and 4xcmp (8 ul cmp).

Wednesday 18th September

Carried out miniprep of 16 MMP9 glycerol stocks candidates which were inoculated overnight the day before; another miniprep was set for 2 samples from zeocin ligation 1 (prepared the day before) which was inoculated overnight (10 ml LB and 40 ul cmp). Nanodrop results of the above

Tube label	ng/ul	260/280
1	97.6	1.85
2	151.8	1.70
4	121.8	1.80
5	103.6/td>	1.83
6	104.8	1.92/td>
7	409.2	1.82
8	220.3	1.79
9	187.3	1.80
10	188.2	1.91
11	82.8/td>	1.88
12	79.5	1.76
13	170.6/td>	1.73
14	117.1	1.92
15	177.8	1.89
17	119.7	1.85
18	167.5	1.69
Zeo lig1A	419.6	1.94
Zeo lig1B	97.2	2.03

Casey single digests recipes

Gel loading order wells: 1x, 1E, 1P, 1u, 13x, 13E, 13P, 13u, 21x, 21E, 21P, 21u, 1.15x, 1.15E, 1.15P, 1.15u, 5.1x, 5.1E, 5.1P, 5.1u.

Carried out gel extraction and purification of Weiling’s MMP9 amplification; total material loaded: 2x50 ul vials from past PCR and 6x50 ul PCR left at 4oC overnight on 17/09/13. After the purification procedure, the concentration of MMP9 was found to be 111 ng/ul and 260/280 indices = 2.13.

Prep digest of purified MMP9 with Dpn1, EcoR1 and Pst1

Component	Xba1 digest (ul)	EcoR1 digest (ul)	Pst1 digest (ul)
DNA	5	5	5
Xba1	1	-	-
EcoR1	-	1	-
Pst1	-	-	1
Buffer 4/3	1 (buffer 5)	1 (EcoR1 buffer)	1 (buffer 3)
BSA	0.5	0.5	0.5
dH2O	2.5	2.5	2.5
Total	10	10	10

Components	Volumes (ul)
DNA	58
Pst1	7
Ecor1	7
Dpn1	7
Buffer 2	10
BSA	5
dH2O	6
Total	100

These were incubated for 2 hours at 37C. This was followed be a PCR purification.

Prep digest of pSB1C3 (the entire stock) with Dpn1, EcoR1, Pst1

Components	Volumes (ul)
DNA	70
Pst1	6
Ecor1	6
Dpn1	6
Buffer 2	10
BSA	2
dH2O	0
Total	100

The digestion was incubated for 2 hours and then incubated for 20 min.

Thursday 19th September

Focus on MMP-9 - Minipreped samples from the 18 inoculations, only 16 of these had growth. - Took the 16 inoculations forward to minipreping. - Made glycerol stocks of the 16 inoculations. - Took nanodrop readings (range varied between 6 ng/ul -45 ng/ul). - Did an analytical digest with Xba1 using 5 samples of the highest concentrations (2, 4, 10, 14, 17). - Gel result didn’t show bands for 4, 2, 14, 17, only sample 10 showed visible bands for both cut and uncut. - Re-inoculated 16 samples from glycerol stocks for 16 hours to for minipreps the following day. - re-PCRed 6 tubes of MMP-9 with MMP9 4 bb RseFW primes (total of 8 tubes to gel extract and purify the following day). Nanodrop of PCR purified and E+P+D digest PSBIC3 (2013 High school iGEM )and MMP9 insert

Tube	ng/ul	260/280
MMP9	184.4	1.87
pSB1C3	27.4	1.73

Xba1 restriction sites in PSBIC3 – 1 restriction site

the recombinant plasmid (with zeocin as an insert) is about 3-6-3.8 kb when cut with Xba1 or EcoR1. Instead the gel run for the xba1 digests have a strong 2 kb band in all the cuts. Possibly, some plasmids may have ligated to themselves.

Repeat digest of minipreps of AA1 ligations (transformation of only using EcoR1)

Samples digested (9 in total): AA1 miniprep col 1à 8 & AA1 5xcmp miniprep 10µl reaction volume

Components	Volumes (ul)
DNA	5
EcoR1	1
Buffer 3	1
BSA	0.5
dH2O	2.5
Total	10

Re-Inocubations from glycerol stocks from both batches of transformation with AA1 ligation pick using: - 2ml LB - 8µl amp - 10µl glycerol stock - Control pSecTag 2A in 8 ul of amp instead of cmp

Glycerol stocks inoculated: 1.2, 1.6, 1.4, 1.18 (from second inoculation, in 4xcmp LB ) All Batch I (from first inoculation, in 1xcmp for col.1-8 and 4xcmp for AA1 5xcmp) &Psectag 2A (Amp).

Weiling: combined MMP-9 minipreps into one tube 11, 12, 13, 14, 17, 18, 1, 2, 4, 5, 6, 7, 10. This was labeled MMP9bbP00L, date, initials.

Nanodrop of pooled	ng/ul	260/280
MMP9 sample	NO DATA	NO DATA
MMP9 bb P00L	169.8	1.91

Saturday 21st September

Minipred-ed 10 ml LB 4x CMP ZEC BB Sample 1 (2z.1) and 6 (7z.2) (from second attempt of zeocin and backbone ligation)

Tube no.	ng/ul	Absorption	260/280
1	3.2	0.087	2.41
26	2.9	0.068	2.51
15	20.1	1.249	1.70
18	21.1	0.590	1.48
21	8.3	0.186	1.20
28	8.4	0.178	1.32

Pooled all 6 samples + Yanika’s minipreped sample IA+IB

	ng/ul	Abs	260/280
Sample	12.3	0.337	1.82

Inoculated 150µl of ZEC BB 1 into 4x cmp and 150 ml LB broth overnight.

Sunday 22nd September

Nanodrop readings (after maxi-prep) of Zec sample 1, ng/ul = 40.7, 260/280 = 1.90