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Revision as of 11:11, 14 October 2014

April

1st

Organization

where to get:

key cards and keys for the lab
cooled centifuge
space in -80 °C
bottles for 70 % ethanol
... (problems of a first year team ^^)

12th

Chemically competent NEB Top10, DH5α and BL21 were made

15th

800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
The efficiency of our competent cells was tested
- Colonies on the agar plates were counted after transformation with 1 µl DNA (147 ng/µl)
- Cells had been incubated in SOC or LB medium

Dilution	200 µl (stock)	100 µl (stock)	1:5	1:10	1:100
DH5α	SOC: 30 LB: 10	SOC: 7 LB: 1	SOC: 1 LB: 2	SOC: 2 LB: 1
NEB Top 10	SOC: 170 LB: 135	SOC: 100 LB: 79	SOC: 25 LB: 22	SOC: 9 LB: 9	SOC: 1 LB:0

BL21 cells were centrifuged and plated; only 128 colonies grew in total indicating a very bad efficiency (~482.11).

Following formula was used to calculate the efficiency:

Efficiency = (CFU /µg DNA) / Dilution

for DH5α: medial efficiency in SOC: 1864.36, and in LB: 440.68
for NEB: medial efficiency in SOC: 6779.66, and in LB: 4698.30
Higher efficiency when using SOC!

22th

Pcq lab PCR advanced primus 25, 96

	Lenght [bp]	% GC	T_m	T_A
EYFP_RFC25	778	61	84	56
REACh1_C	481	62	84	57
REACh1_N	320	59	82	54
REACh2_C	487	62	83	57
REACh2_N	320	59	82	54

Program name IGEM.cyc

Parameter	Duration	Temp [°C]
Denature	10:00	98
Denature	00:30	98
Anneal	00:30	52
Elongate	02:36	72
Elongate	05:00	72
Store	forever	8

RFC25_F RFC25_R
RFC25_F REACh1_R
REACh1_F RFC25_R
RFC25_F REACh2_R
REACh2_F RFC25_R

23th

5 µl of the PCR products were run on a 1,5% agarose gel
DNA bands were purified from gel with the High Pure Product Purification Kit. Full length products were contaminated with REACh1

SOE
ca. 30 ng/µL

	20x	-> 20 µL thereof: 30x
HF	10	10
dNTP	4	4
Template	2x 1	10
Phusion	0.5	0.5
Primer	-	2x 2.5
Water	33.5	21.5

Anneling: 52 °C

Elongation: 20 sec

PCR with Phusion polymerase

	Volume [µL]
HF	10
dNTP	4
Template	1
Phusion	0.5
Primer	2x 2.5
Water	29.5
Total	50

Digestion for restriction with NEB enzymes

	Volume
Destination Vector	500 ng
EcoRI-HF	1 µL
PstI	1 µL
10x NEB Buffer 2.1	5 µL
Water	??? µL
Total	??? µL

ligation with NEB Kit

28th

The DNA concentration of the SOE2 products were measured after purification
1: 57 ng/µL
2: 69.5 ng/µL

29th

PCR SOE1 and SOE2

30th

A PAGE of the SOE2 products was run on agarose gel for checking
→ no positive results; only full length products were amplified

SOE PCRs were run again
→ more template
→ hot start
→ faster
→ elongation: 15 min
→ 20 cycles

SOE1

Template A+B → 20 µL → 13.8 + 6.2 5.3 + 14.7

	volume [µL]
HF	10
dNTP	4
Template	20
Phusion	0.5
Water	13.5

SOE2

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Team:Aachen/Notebook/Wetlab/April