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From 2012.igem.org

Weeks:

June			July				August				September				October				November

Week 4: 2nd July - 8th July

Monday, 2nd July

1. T10 bacteria transformation with DNA ligated on Friday (29/06)- protocol as before
2. Transformed bacteria spreaded on Petri's plate- LB-agar-ampiciline- incubation 37 degrees Celsius, overnight
3. SOC preparation:
   • 1,4g Hanahan's Broth
   • 0,18g Glucose
   • 50mL MiliQ water

filtrated on microfilter (if you have more important volume, autoclave it)

Tuesday, 3rd July

1. Designing starters
2. Weekly team meeting- discussion about work organization

Wednesday, 4th July

1. Gel electrophoresis of DNA digests (from 28.06.) followed by cutting-of bands of interests (pCS2+ plasmid and XFP plasmids)
2. Gel-extraction (Kit used: QIA quick gel extraction) followed by nano-drop measurements in order to get information about DNA concentration.
   Result: very low quantity of DNA. We realized that quantity of DNA used for digestion was too low (less than 2 micrograms of DNA/sample).
3. DNA Digestion (of pCS2+, CFP, RFP, GFP, YFP). Protocol(Final volume of sample: 30 uL):
   • 3 uL Buffer 10x (choose a right buffer for each enzyme you use)
   • 1 uL Restriction Enzyme 1
   • 1 uL Restriction Enzyme 2
   • 2 ug DNA (calculate the volume which you need to add, depends of the DNA concentration)
   • c uL ddH2O (fill till 30 uL)
   • Incubation 3h at 37 degrees

Thursday, 5th July

Gel Extraction 1:

1. Excise the DNA fragment from agarose gel
2. Weight the gel slice
3. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg = 100 uL)

   Type	Gel weight (g)	V QG (uL)	V iso (uL)
   pCS2	0,13	390	130
   GFP	0,10	300	100
   YFP	0,08	240	80
   CFP	0,10	300	100

4. Incubate at 50 degrees Celsius for 10 min to dissolve the gel
5. Add 1 gel volume of isopropanol to the sample and mix
6. Apply the sample to the QIAquick column to bond DNA
7. Centrifuge at 13 000 rpm for 1 min and discard flow-through
8. Add 500 uL of Buffer QG to the column
9. Centrifuge at 13 000 rpm for 1 min and discard flow-through
10. To wash, add 750 uL of Buffer PE
11. Centrifuge at 13 000 rpm for 1 min and discard flow-through
12. Centrifuge the column again at 13 000 rpm for 1 min and place the column in a clean 1.5 mL tube
13. Add 50 uL of Buffer EB to elute DNA
14. Check DNA concentration with Nanodrop

DNA Concentration

Type	Concentration (ng.uL-1)
pCS2	9,4
GFP	3,1
YFP	4,7
CFP	4,7

But: Concentrations are really low => Need to optimize the protocol and make a new gel migration.

Gel Migration:

• Gel at 0,08%
• V tae = 30 mL
• m aga = 0,24 g
• V bet = 3 uL

No Band for RFP

Gel Extraction 2

Type	Gel weight (g)	V QG (uL)	V iso (uL)
pCS2	0,084	252	84
GFP	0,040	120	40
YFP	0,023	69	23
CFP	0,050	150	50

Protocole modification: Elution in two times 25 uL with water RNAse free instead of EB buffer.