IGEM:MIT/2007/Notebook/2007-7-9

Nanodrop of Purified pSB3k3 from Mini-Prep of DB3.1 LQ

1. 38.9 ng/µl

2. 38.7 ng/µl

3. 33.7 ng/µl

4. 33.8 ng/µl

Digestion of Purified pSB3k3 from this morning

Digestion 1:

25.6 µl pSB3k3 (38.9 ng/µl)

0.5 µl BSA

5.0 µl NEB3 Buffer

1.0 µl EcoRI

1.0 µl PstI

16.9 µl H2O

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50µl Total

Digestion 2:

25.8 µl pSB3k3 (38.7 ng/µl)

0.5 µl BSA

5.0 µl NEB3 Buffer

1.0 µl EcoRI

1.0 µl PstI

16.7 µlet

Latex cheat sheet. H2O

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50µl Total

• Placed in 37C room at 4:30pm

Liquid Culture of DB3.1

Took two colonies from each plate (1A, 1B, 2A, 2B, 3A, 3B)

• Placed in 37C room for overnight growth

Poured Plates

• 16 LB+Amp (180µl in 450ml LB)
• 28 LB+Kan (475µl in 475ml LB)

Purification of pSB3k3, F2620 and B0034 Digestion

pSB3k3: 3.5 ng/µl F2620: 17.7 ng/µl B0034: 19.9 ng/µl

• Because B0034 is under 200bp, we had to use a different kit (Qiagen Nucleotide Removal Kit)
• For pSB3k3 and F2620, we used the Qiagen PCR Purification Kit

Ligation of F2620 and B0034 into pSB3k3

[math]\displaystyle{ Mass_{insert}=4\frac{length_{insert}}{length_{backbone}}M_{backbone} }[/math]

Nanodrop of parts:

• pSB3k3----3.5 ng/ul
• B0034----19.9 ug/ul
• F2620----17.7 ug/ul

Calculation

[math]\displaystyle{ \frac{50 ng}{3.5 ng/ \mu l}=14.3 \mu l }[/math] pSB3K3

[math]\displaystyle{ 17.7x = 4\frac{1070}{2750}50 }[/math]

[math]\displaystyle{ x=4.4\mu l }[/math]F2620

[math]\displaystyle{ 19.9 x = 4 \frac{17}{2750} 50 }[/math]

[math]\displaystyle{ x=.062 \mu l }[/math] B0034

Reaction Mix

'Note: In general the higher volume we make, the worse the transformation efficiency'

• 87.5 ng/ 25 ul -> Backbone
• 12.5 ul -> 44 ng
• 6 ul B0034 -> 100 ng
• 6 ul F2020 -> 100 ng
• 3 ul ligase -> 100 ng
• 3 ul ligase buffer
• .5 ul ligase
• 2 ul H2O
• ----
• 30 ul (Ideally 20 ul or 10 ul) Room Temp, 90 min

Transformation

• XL-1 Strain

1. Aliquot 100 ul cells
2. Add 1.7 ul B-mercaptoethanol
3. Leave on ice for 10 min, swirling occasionally
4. Add 2 ul DNA
5. Ice for 30 min
6. Heat pulse for 45 seconds at 42 degrees C (critical)
7. Ice for 2 min
8. Add 400 ul 42 degrees C heated LB
9. 37 degree C room shaker 220 rpm for 1 hour

DH5alpha strain

1. Aliquot 100 ul cells
2. Add 2 ul DNA
3. Ice for 30 min
4. Heat pulse for 30 seconds
5. Ice 2 min
6. Add 1 ml 42 degrees C LB
7. 37 degree room shaker 229 rpm for 1 hour

Plating

1. Amp xL-1 20 ul
2. Amp xL-1 200 ul
3. Kan xL-1 20 ul
4. Kan xL-1 200 ul
5. Amp DH5alpha 100 ul
6. Amp DH5alpha 200 ul
7. Kan DH5alpha 100 ul
8. Kan DH5alpha 300 ul