Team:Dundee/lucylabjournal

LABJOURNAL

BioSpray

Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.

Chromium Detector

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Fingerprint Aging

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Week Beginning 1/6/2015

Summary

Lactoferrin binding protein A (LbpA) was successfully cloned into the vector pSB1C3.

1/6: Amplification of LbpA

Aim of Experiment: Amplify LbpA gene from N.meningitidis template.

Protocols Used: Click here to see our PCR protocol.

Results: Figure 1

Next Steps: Since the LbpA gene was amplified successfully, it was ready to be digested for subsequent cloning into pSB1C3.

2/6: Restriction Digests and Ligations

Aim of experiment: To digest LbpA with BamHI and EcoRI and then ligate it into pSB1C3.

Protocols Used: Restriction Digests Ligations

Results: N/A

Next Steps: Ligations of LbpA + pSB1C3 were left overnight until the next day when they would be transformed into JM110 cells.

3/6: Transformations of LpbA + pSB1C3 into JM110

Aim of experiment: To transform the pSB1C3 + LbpA ligations into JM110 cells.

Protocols Used: Transformations

Results: N/A

Next Steps: Transformations will be checked tomorrow and if successful, overnight cultures will be set up.

4/6: Overnight Cultures

Aim of experiment: To set up overnight cultures of the LbpA + pSB1C3 JM110 colonies.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: A miniprep will be performed on the overnight cultures and a pre-sequence digest test performed tomorrow.

5/6: Plasmid Purification and Pre-Sequencing Digest Check

Aim of experiment: To miniprep the LbpA + pSB1C3 JM110 overnight cultures and perform a pre-sequence restriction digest to check for the presence of the insert.

Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Restriction Digests

Results: N/A

Next Steps: Since the pre-sequencing digest worked, the miniprep will be sent for sequencing.

Week 2

Week summary

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8/6: Amplification of LbpA for Cloning into pQE80-L

Aim of experiment: To amplify LbpA for subsequent cloninng into the pQE80-L vector.

Protocols Used: PCR Protocol

faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl.

Day 2

Aim of experiment: ultricies nec tellus eu

Protocols Used:

faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl.

Day 3

Aim of experiment: ultricies nec tellus eu

Protocols Used:

faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl.

Day 4

Aim of experiment: ultricies nec tellus eu

Protocols Used:

faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl.

Day 5

Aim of experiment: ultricies nec tellus eu

Protocols Used:

faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl. Morbi felis lectus, ultricies nec tellus eu, faucibus eleifend lectus. In molestie augue leo, id imperdiet ante imperdiet at. Quisque ultrices neque sit amet felis tincidunt tristique at sed nisi. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos. Ut suscipit ut massa in ultricies. Nunc nec tincidunt nunc, ut consectetur lorem. Sed non justo nislo nislo nisl.

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PCR Protocol

PCR reaction mixtures were set up according to the table below. The length of all sequences used is stated in the Sequences section.

Reactants	Volume (µl)
DNA	1
DMSO	2.5
Forward Primer	0.5
Reverse Primer	0.5
Herculase Buffer	10
dNTP	0.5
Herculase	0.5
H2O	34.5

**PCR Programme**

Elongation time was altered according to the length of the sequences.

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Restriction Digest Protocol

Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Journal section.

	Post Plasmid Purification	Post PCR	gBlock
DNA	1mg	50µl	100ng
Cutsmart Buffer (µl)	6	6	6
Water (µl)	As required	1	As required
Enzyme 1 (µl)	1.5	1.5	1.5
Enzyme 2 (µl)	1.5	1.5	1.5
Total Volume (µl)	30	60	30

The digests were incubated at 37^oC for 3 hours.
Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer was added at the 2 hour and 2.5 hour mark.

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Ligation Protocol

Ligation reactions were set up according to the table below. The vector to insert ratio is stated for each specific reaction in the Journal section.

	Vector (µl)	2:1 (Vector: Insert) [µl]	3:1 (Vector: Insert) [µl]	4:1 (Vector: Insert) [µl]
T4 DNA Ligase	1	1	1	1
Ligase Buffer	1	1	1	1
Vector	1	1	1	1
Insert	-	2	3	4
H20	7	5	4	3

The reactions were incubated at room temperature for at least 3 hours.

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Transformation Protocol

1. 100µl of competent cells were defrosted. Thaw on ice.

2. Agar plates were taken out of 4^oC to warm to room temperature.

3. 1-5µl of DNA was added to cells.

4. Cell and DNA mixture was left on ice for 20 minutes.

5. Mixture was placed in 42^oC water bath for 90 seconds.

6. Mixture was put back on ice for 2 minutes.

7. 1ml of LB (without antibiotic) was added to cells.

8. Cells were allowed to grow for 1 hour at 37^oC in a shaking incubator.

9. Cells were then spun down and the supernatant discarded.

10. The pellet was resuspended in the 100µl.

Cells were plated on agar plates with appropriate antibiotic.

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Figure 1: LbpA PCR. The image shows the gel ran of the LbpA PCR product. The expected size of the fragment was 2832bp which corresponds to the observed band on the gel.

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