# SBB09Ntbk-Dirkvs

(Difference between revisions)
 Revision as of 15:37, 27 April 2009 (view source) (→Dirk 14:02, 27 April 2009 (EDT))← Previous diff Current revision (15:45, 27 April 2009) (view source) (→Running the assay on cells) Line 58: Line 58: * Resuspend cells * Resuspend cells * Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube * Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube - * Incubate overnight at 37C + * Incubate in rocker overnight at room temperature ==== Determining the AG4 absorption wavelengths ==== ==== Determining the AG4 absorption wavelengths ====

## Dirk 14:02, 27 April 2009 (EDT)

```Summary of Activities:
* Over the weekend, the TBS+AgNO3 assays yielded colour for samples 12, 13, 14, 15
* Prepared a repeat of TBS+AgNO3 assay, and left overnight - labelled AG4 Assay V2
* The absorbance readings were inconclusive - the samples looked like the control (probably due to the large presence of cells)
To do today:
* Prepare a second round of TBS+AgNO3 assay
* Find the absorption wavelengths for the AG4 assay
* Quantify the first round of results for the AG4 assay
```

### AG4 Silver biomineralization assay

#### Preparation of AgNO3 solution

• Weigh AgNO3 powder in an Eppendorf tube (weighed 0.088 grams)
• AgNO3 has MW 169.87 g/mol - dilute 0.088g with 0.500ml for ~1 M solution
• Make 500ul of 10mM solution by adding 5ul of 1 M AgNO3 into 495ul of H2O
• Make 200ul of 1mM solutions by adding 20ul of 10mM AgNO3 into 180ul of H2O

#### Preparation of cells for assay

• Spin down 2ml of cells and throw away supernatant
• Add 200 ul of TBS and resuspend cells
• Spin down the cells and throw away supernatant

#### Running the assay on cells

• Add 180 ul of TBS to each pellet (prepared above)
• Resuspend cells
• Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube
• Incubate in rocker overnight at room temperature

#### Determining the AG4 absorption wavelengths

In order to get quantitative data, we need to find out which wavelengths are absorbed by the AG4 peptide silver precipitate. We chose a sample with good colour, and scanned it through the colour spectrum to find out the absorption wavelengths compared to the control sample. The instrument used was the Tecan spectrophotometer, and measurements were taken by Susan, Roger, Carlos, CA.

• Results were inconclusive
• Control and samples had very similar absorption spectra
• Results probably due to presence of cells
• We are considering how to isolate the silver from the rest of the sample (mechanical lysis and separation)

## Dirk 13:20, 22 April 2009 (EDT)

```Summary of Activities:
* Prepared Tris buffer and AgNO3 solutions
* Incubated cells 11-17 + Control in LBTAg media
* Incubated cells 11-17 + Control in TBS + AgNO3 for silver binding assay
To do today:
* Prepare Tris buffer solution
* Prepare AgNO3 solution
* Prepare LB + Tris + AgNO3 growing media (LBTAg)
* Incubate and grow cells in LBTAg media
* Run silver binding assay
```

### AG4 Silver biomineralization assay

#### Preparation of Tris buffer solution

• Add 605 mg Tris and 876 mg NaCl to 80 ml H2O
• Adjust pH to 7.4 by adding HCl and bring volume to 100 ml

#### Preparation of AgNO3 solution

• Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams)
• AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution
• Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O

#### Preparation of cells for assay

• Spin down 2ml of cells and throw away supernatant
• Add 200 ul of TBS and resuspend cells
• Spin down the cells and throw away supernatant

#### Preparing cells for growth in LBTAg media

• Dispense 3ml of LB media into 9 chambers in growth rack
• Add 200 ul of Tris buffer solution to the LB media
• Add 32 ul of 10 mM AgNO3 to make 0.1 mM LBTAg media
• Pick cells from each washed pellet (above) and add to corresponding growth chambers
• 11-17 + Control + another one of 11
• Add 3ul of 1000x Arabinose to the 9th chamber containing a second culture of cells 11
• Cover the rack and incubate overnight at 37C

#### Running the assay on cells

• Add 180 ul of TBS to each pellet (prepared above)
• Resuspend cells
• Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube
• Add 2ul of 100x arabinose
• Incubate overnight at 37C

## Dirk 5:04, 21 March 2009 (PST)

I was not in lab today, but Susan and Roger carried out the following work:

```Summary of Activities:
* Grew one colony from each sample (11-17) and control in 4ml LB media (with CA)
* Grew one colony from each sample (11-17) and control in 4ml LB media and 0.1mM AgNO3 (with CA)
* [http://openwetware.org/wiki/Image:Silver_binding1.doc growing cells grid]
```

## Dirk 13:41, 20 April 2009 (EDT)

The composite parts have been assembled, and now we are doing assays for the different constructs. I am doing the AG4 silver binding peptide assay (Construct 18) with Carlos, Roger, Sadao, and Susan.

Our assay protocol can be found here:

```Summary of Activities:
* Tested 1M, 100mM, 10mM, 1mM, 0.5mM, 0.1mM AgNO3 in LB media
* A precipitate formed with AgNO3 in LB all the way down to 0.5mM
* 0.1mM AgNO3 in LB media did not form a visible precipitate
* Transformed constructs into bacteria (Roger and Susan)
* Plated the cells (Roger and Susan)
* We will use constructs 11-17 and a pBca9145-Bca1363 as control
To do today:
* Test AgNO3 precipitation in LB Media
```

### AG4 Silver biomineralization assay

#### Preparation of AgNO3 solutions

• Weigh AgNO3 powder in an Eppendorf tube (weighed 0.88 grams)
• AgNO3 has MW 169.87 g/mol - dilute 0.88g with 0.5ml for ~10 M solution
• Dilute it down to 10mM
• Create 1 mM, 0.1 mM, and 0.05 mM dilutions by adding 9+1, 9+1, and 1+1 parts of H2O and AgNO3 solution

#### Test AgNO3 precipitation in LB media

• Prepare Eppendorf tubes with 200ul of LB media
• Dilute 1M, 100mM, 10mM, 1mM, 0.5mM, and 0.1mM AgNO3 in LB media
• Mix and check for precipitate - if no precipitate, spin down in centrifuge and look for pellet

## PROJECT STATUS SUMMARY - Dirk 16:08, 18 March 2009 (EDT)

### Construct 17 M10022 {<Beta Roll>}

4 clones submitted for sequencing (sequence numbers sbb090-sbb093)

### Construct 18 M10023 {<AG4>}

Part complete (by CA)

Part complete

## Dirk 14:26, 18 March 2009 (EDT)

```Summary of Activities:
* Construct 17 was cloned and grown by CA, and handed back to me for miniprepping
* Miniprep of 4 clones of Construct 17 was carried out
* Miniprepped clones were stored in 48 ul volumes, labelled B-Roll Clone 1-4 18.03.2009 MINIPREPPED
* Mapping of all 4 clones of Construct 17 came out well - see picture below
* All 4 clones of Construct 17 M10022 {<Beta Roll>} were submitted for sequencing (read numbers sbb090-sbb093)
* CA confirmed that Construct 18 M10023 {<AG4>} is complete and sequenced
To do today:
* Miniprep and mapping of 4 clones of Construct 17
* Submit Construct 17 for sequencing
```

### Construct 17 {<Beta Roll>}

Miniprep purification of Construct 17 pBca9495AK-M10022 DNA

• MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
1. Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10. Wash each column with 500 uL of PB buffer.
11. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
12. Wash with 750uL of PE buffer (washes the salts off the resins).
13. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
14. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
15. Label new tubes and put columns in them.
16. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
17. Spin in centrifuge at top speed for 30 seconds.
18. Take out columns and cap the tubes.
19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

Mapping of Construct 17 pBca9495AK-M10022

Mapping for Construct 17

Set up the following 10uL reaction in a PCR tube:

```6.5 uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)
```
• Incubate at 37 on the thermocycler for 30 minutes
• Run an analytical gel
• Take a picture of the gel
• Calculate the expected fragment sizes
• Are the calculated sizes consistent with the bands on the gel?

Sequencing of Construct 17

• Clone 1 of Construct 17 was sent for sequencing. The read number is sbb090
• Clone 2 of Construct 17 was sent for sequencing. The read number is sbb091
• Clone 3 of Construct 17 was sent for sequencing. The read number is sbb092
• Clone 4 of Construct 17 was sent for sequencing. The read number is sbb093

### Construct 18 M10023 {<AG4>}

• CA confirmed that Construct 18 is complete and has been sequenced
• The sequence came out perfect, and can be found at sbb063

## Dirk 17:40, 16 March 2009 (EDT)

```Summary of Activities:
* Construct 19 Clone 1 sequence turned out perfect
* Results entered into spreadsheet and wiki
To do today:
* Analyse the sequence for Construct 19 Clone 1
* Enter sequence analysis and part summary for Construct 19 into Wiki and spreadsheets
```

### Construct 19 {<CBK>}

Sequencing Analysis

• The sequence matched perfectly for the region between the EcoRI and BamHI restriction sites
• There was one point mutation within the origin of replication (base 860 is an A instead of a G)
• Sequence entered into sbb039

## Dirk 13:24, 9 March 2009 (EDT)

```Summary of Activities:
* No work on constructs 17 or 18 carried out
* Transformation of Construct 19 was good - TA grew 2 clones for miniprep and mapping
* Miniprep of Construct 19 in pBca9495AK was carried out
* Clone 1 of Construct 19 was sent for sequencing - the read number is [[SBB09sbb/sbb039 | sbb039]]
* Clone 2 of Construct 19 MINIPREPPED was stored in the blue-stripes box, with volume 41.5 ul
* Mapping of Construct 19 clones 1 & 2 was carried out - results are good (no picture taken)
To do today:
* Miniprep and mapping of Construct 19 clones
```

### Construct 19 {<CBK>}

Miniprep purification of Construct 19 pBca9495AK-M10046 DNA

• MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
1. Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10. Wash each column with 500 uL of PB buffer.
11. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
12. Wash with 750uL of PE buffer (washes the salts off the resins).
13. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
14. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
15. Label new tubes and put columns in them.
16. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
17. Spin in centrifuge at top speed for 30 seconds.
18. Take out columns and cap the tubes.
19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

Mapping of Construct 19 pBca9495AK-M10046

Set up the following 10uL reaction in a PCR tube:

```6.5 uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)
```
• Incubate at 37 on the thermocycler for 30 minutes
• Run an analytical gel
• Take a picture of the gel
• Calculate the expected fragment sizes
• Are the calculated sizes consistent with the bands on the gel?

Sequencing of Construct 19

• Clone 1 of Construct 19 was sent for sequencing. The read number is sbb039

## Dirk 13:13, 6 March 2009 (EST)

```Summary of Activities:
* Transformation of Construct 17 looked pretty decent - Tim grew up 4 clones for miniprep and mapping
* Miniprep of Construct 17 carried out by CA
* Construct 18 colonies didn't grow. The construct was abandoned (Although CA might try to rescue it)
* Ligation of Construct 19 into pBca9495AK was carried out
* Construct 19 GP CLEAN (pre-ligation) stored in blue-stripes box with less than 10 ul volume
* Carried out transformation of Construct 19 pBca9495AK into competent cells
* Carried out a rescue of Construct 19 cells
* Construct 19 cells were plated onto an AK dish and left do incubate in room 327
To do today:
* Miniprep and map of Construct 17
* Ligation (into pBca9495AK), transformation, and plating of Construct 19
```

### Construct 17 {<Beta Roll>}

Miniprep purification of Construct 17 in pDrive DNA - Carried out by CA ---Dirk 14:09, 6 March 2009 (EST)

• MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
1. Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10. Wash each column with 500 uL of PB buffer.
11. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
12. Wash with 750uL of PE buffer (washes the salts off the resins).
13. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
14. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
15. Label new tubes and put columns in them.
16. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
17. Spin in centrifuge at top speed for 30 seconds.
18. Take out columns and cap the tubes.
19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

### Construct 18 {<AG4>}

Construct 18 was abandoned - the colonies didn't grow, and it is too late to start over. Dirk 13:26, 6 March 2009 (EST)

### Construct 19 {<CBK>}

Ligation (into pBca9495AK), transformation, and plating

Ligation of Construct 19 EcoRI/BamHI digests into pBca9495AK

• Set up the following reaction:
``` 6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase
```
• Pound upside down on the bench to mix
• Give it a quick spin to send it back to the bottom of the tube
• Incubate on the benchtop for 30min
• Put on ice and proceed to the transformation

Transformation of Construct 19 and pBca9495AK cloning vector by heat-shock

1. Put your ligation mixture on ice, let it cool a minute or two
2. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
3. Let sit on ice for 10 min
4. Heat shock for 2 min at 42
5. Put back on ice for 1 min
6. For ampicillin selection, you can plate immediately, otherwise:
7. Add 100uL of 2YT, let shake in the 37 degree incubator for 60 min

## Dirk 17:04, 4 March 2009 (EST)

```Summary of Activities:
* Construct 17 cells were plated on AMP dish and left to incubate in room 327
* Construct 18 cells were plated on AK dish and left to incubate in room 327
To do today:
* Plate Construct 17 cells on AMP dish
* Plate Construct 18 cells on AK dish
```

### Construct 17 {<Beta Roll>}

Plate Construct 17 cells on AMP dish

1. Plate on selective antibiotics, let incubate overnight

### Construct 18 {<AG4>}

Plate Construct 18 cells on AK dish

1. Plate on selective antibiotics, let incubate overnight

## Dirk 13:18, 4 March 2009 (EST)

```Summary of Activities:
* Carried out ligation of Construct 17 into vector pDrive
* Carried out transformation of Construct 17 - must return in 40 mins to plate cells
* Construct 17 Broll PCR PROD stored in blue-striped box
* Carried out ligation of Construct 18 into vector pBca9495AK
* Carried out transformation of Construct 18 - must return in 40 mins to plate cells
* Construct 18 DIGESTED CLEAN stored in blue-striped box with 49 ul volume
* Carried out gel purification of Construct 19 - bands were difficult to see
* Carried out GP Zymo Cleanup of Construct 19 GP product
* Did not carry out ligation of Construct 19
* Construct 19 GP CLEAN stored in blue-stripes box with 8.5 ul volume
To do today:
* Ligation of Construct 17 in preparation for cloning and sequencing, before making into a part
* Ligation of Construct 18
* Gel purification and ligation of Construct 19
```

### Construct 17 {<Beta Roll>}

Ligation of Construct 17 PCR product into pDrive Cloning Vector

• Add 1 ul of pDrive cloning vector (50 ng/ul)
• Add 1 ul of Construct 17 PCR product
• Add 3 ul of RNase-free water
• Add 5 ul of Ligation Master Mix, 2x
• Mix the ligation reaction and incubate for 30 min at room temperature

Transformation of Construct 17 and pDrive cloning vector by heat-shock

1. Put your ligation mixture on ice, let it cool a minute or two
2. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
3. Let sit on ice for 10 min
4. Heat shock for 2 min at 42
5. Put back on ice for 1 min
6. For ampicillin selection, you can plate immediately, otherwise:
7. Add 100uL of 2YT, let shake in the 37 degree incubator for 60 min

### Construct 18 {<AG4>}

Ligation of Construct 18 EcoRI/BamHI digests into pBca9495AK

• Set up the following reaction:
``` 6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase
```
• Pound upside down on the bench to mix
• Give it a quick spin to send it back to the bottom of the tube
• Incubate on the benchtop for 30min
• Put on ice and proceed to the transformation

Transformation of Construct 18 and pBca9495AK cloning vector by heat-shock

1. Put your ligation mixture on ice, let it cool a minute or two
2. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
3. Let sit on ice for 10 min
4. Heat shock for 2 min at 42
5. Put back on ice for 1 min
6. For ampicillin selection, you can plate immediately, otherwise:
7. Add 100uL of 2YT, let shake in the 37 degree incubator for 60 min

### Construct 19 {<CBK>}

Gel purification of Construct 19 digestion product

• Load 10uL of digestion product premixed with 2uL of loading buffer in a single well of a 1% agarose gel
• Cut out the bands, put them into a single 1.5mL microcentrifuge tube
• Proceed with the Zymo Gel Purification procedure

Zymo Gel Purification of Construct 19 digestion gel purification product

• All spins until the drying step are 15 second full speed spins.
1. cut out bands minimizing extra gel matter.
2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
3. heat at 55, shake and/or vortex until the gel has dissolved.
4. If the DNA is <300bp add 250uL of isopropanol
5. transfer into the Zymo column inside a collection tube (small clear guys)
7. Add 200 uL of PE buffer (which is basically 70% ethanol)
9. Add 200 uL of PE buffer
11. spin for 90 seconds, full speed to dry.
12. elute with 8.5 uL of water into a fresh Eppendorf tube

## Dirk 13:18, 2 March 2009 (EST)

```Summary of Activities:
* Construct 17 PCR reactions worked, as verified by Chris Anderson
* New oligos for a second restriction site in Construct 17 were ordered
* No work on Construct 17 was carried out today
* First Zymo small fragment clean up of Construct 18 wobble product carried out
* Digestion of Construct 18 was carried out (with full wobble product volume - 50 ul)
* Second Zymo small fragment clean up of Construct 18 digestion product carried out
* Construct 18 DIGESTED CLEAN stored in blue-stripes box with 50 ul
* Digestion of Construct 19 was carried out (with 8 ul of gene synthesis reaction volume)
* Small fragment Zymo clean up of Construct 19 digestion product carried out
* Gel purification of Construct 19 digestion product not carried out
* Analytical gel of Construct 19 pre-digestion was carried out - part was there
* Construct 19 DIGESTED CLEAN NOGP stored in blue-stripes box with 10 ul
* Construct 19 PRE DIGEST stored in blue-stripes box with 32 ul left

To do today:
* Clean, digest, clean, and ligation of Construct 18
* Analytical gel of Construct 19 second PCR product
* Digest, clean, gel purification, and ligation of Construct 19
```

### Construct 18 {<AG4>}

Clean, digest, clean, and ligation of Construct 18.

First Small-Frag Zymo Cleanup of Construct 18

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• spin for 90 seconds, full speed to dry.
• elute with water into a fresh Eppendorf tube

EcoRI/BamHI Digest of Construct 18 Wobble Product

For wobble products, you will digest the entire extension reaction-worth of DNA. Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.

• Set up the following reaction in a PCR tube:
• 50uL eluted DNA
• 5.7uL NEB Buffer 2
• 1uL EcoRI
• 1uL BamHI
• Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
• Incubate the reaction at 37 degrees on the thermocycler
• Proceed to another Zymo small fragment cleanup

Second Small-Frag Zymo Cleanup of Construct 18

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• spin for 90 seconds, full speed to dry.
• elute with water into a fresh Eppendorf tube

### Construct 19 {<CBK>}

Analytical gel results for Construct 19

Digest, clean, gel purification, and ligation of Construct 19

EcoRI/BamHI Digest of Construct 19 PCR Products

For PCR products, you will only digest a portion of your purified PCR product. Note that you must make a minor modification to the procedure if your DNA is shorter than 300bp (you add a little isopropanol to the ADB after melting).

• Set up the following reaction:
• 8uL of eluted PCR product
• 1uL of NEB Buffer 2
• 0.5uL EcoRI
• 0.5uL BamHI
• Incubate at 37 degrees on the thermocycler for 1hr
• Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
• If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

Analytical Gel of Construct 19 second PCR product

• Load 3uL of digestion product premixed with 1uL of loading buffer in a single well of a 1% agarose gel
• Results shown to the right

Small-Frag Zymo Cleanup of Construct 19 digestion product for storage before Gel Purification

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• spin for 90 seconds, full speed to dry.
• elute with 10ul water into a fresh Eppendorf tube

## Dirk 15:24, 27 February 2009 (EST) Starting over with Construct 17

```Summary of Activities:
* Fixed errors in construction file
* Found out the correct template DNA (CFT03)
* Started the PCR reaction for Construct 17 A, B, and C using CFT03 as template
** A is the strand upstream from the restriction site (379 bp)
** B is the downstream strand from the restriction site (89 bp)
** C is the full part without restriction site removal (436 bp)
* Need to find out what the exact sequence will be for each of these PCR products

To do today:
* First PCR of Construct 17, making parts A and B (second attempt)

```

### Construct 17 {<Beta Roll>}

Dilute oligonucleotides for construct 17

• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 17 A (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007F (10uM)
• 1 uL Oligo Odvs008rR (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uL Template DNA E.coli 0157:H7
• Run this PCR on program 55

Prepare the PCR reaction for construct 17 B (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007R (10uM)
• 1 uL Oligo Odvs008rF (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uLTemplate DNA E.coli 0157:H7
• Run this PCR on program 55

Prepare the PCR reaction for construct 17 C (Straight forward PCR - no restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007R (10uM)
• 1 uL Oligo Odvs008rF (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uLTemplate DNA E.coli 0157:H7
• Run this PCR on program 55

## Dirk 13:13, 27 February 2009 (EST)

```Summary of Activities:
* Gel purification of Construct 17 parts A and B was attempted - parts weren't there: either PCR failed, or DNA degraded
* Construct 17 parts A and B (pre GP) stored in blue-stripes box, bound in red tape
* Restarted the work with Construct 18
* Wobble PCR reaction for Construct 18 was prepared and given in red tape
* Discarded the Construct 19 (IPA ERROR) container
* No more work was carried out with Construct 19 today (no digestion)
* Construct 19 (PRE DIGEST) stored in blue-stripes box

To do today:
* Second PCR of Construct 17
* Prepare Wobble PCR reactions of Construct 18 (STARTING OVER)
* Clean up, digestion, clean up, and ligation of construct 19 PRE DIGEST (Do GP afterwards - ie, normal PCR digest)

```

### Construct 17 {<ECOHLY>}

Gel purification of Construct 17 parts A and B

• For each PCR, load 10uL of PCR product premixed with 2uL of loading buffer in a single well of a 1% agarose gel
• Cut out the bands, put them into a single 1.5mL microcentrifuge tube
• Proceed with the Zymo Gel Purification procedure

### Construct 18 {<AG4>}

Prepare PCR reaction for construct 18 (Wobble) (Further dilutions not necessary)

• 29 uL water
• 5 uL Expand 10x Buffer 2
• 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• The bottle was mislabelled - used 10 mM dNTPs instead. Dirk 15:06, 27 February 2009 (EST)
• 5 uL Oligo Odvs002F (100uM)
• 5 uL Oligo Odvs002R (100uM)
• 0.75 uL Expand Polymerase 1

### Construct 19 {<CBK>}

No work was carried out with Construct 19 today.

## Dirk 13:31, 25 February 2009 (EST)

```Summary of Activities:
* Second PCR of Construct 17 was not carried out
* First PCR products A+B stored in box, bound in red tape
* Carried out the digestion and second cleaning of Construct 18
* Forgot to add the ADB buffer to the second cleaning of Construct 18 - DNA might be lost
* 50 ul eluted DNA Construct 18 - MAYBE LOST - stored in box
* Did first clean up and digest of Construct 19
* IPA was incorrectly added to the Construct 19 digested DNA - a small fragment clean up was carried out to correct for this mistake
* Gel purification of Construct 19 was not carried out
* 40 ul eluted DNA Construct 19 - PRE DIGESTION - stored in box
* 50 ul eluted DNA Construct 19 - IPA ERROR - stored in box (note that this is diluted 5x)
* Second clean up and ligation of Construct 19 still to be carried out

To do today:
* Second PCR of Construct 17 (Correction: this was not an SOE reaction)
* Finish digestion and cleaning of Construct 18
* Clean up, digest, clean up, and ligation of Construct 19
```

### Construct 17 {<Beta Roll>}

Nothing was carried out with construct 17 today.

### Construct 18 {<AG4>}

Digest, and then cleanup again of construct 18.

EcoRI/BamHI Digest of Construct 18 Wobble Product

• Set up the following reaction in a PCR tube:
• 50uL eluted DNA
• 5.7uL NEB Buffer 2
• 1uL EcoRI
• 1uL BamHI
• Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
• Incubate the reaction at 37 degrees on the thermocycler for 1 hour
• Proceed to another Zymo small fragment cleanup

Second Small-Frag Zymo Cleanup of Construct 18

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Forgot to put the ADB buffer!!! Dirk 15:14, 25 February 2009 (EST)
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• spin for 90 seconds, full speed to dry.
• elute with water into a fresh Eppendorf tube

### Construct 19 {<CBK>}

Clean up and digest of Construct 19

First Small-Frag Zymo Cleanup of Construct 19

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• found out what the blue liquid in the previous wash (construct 18) was: the ethanol dissolved some of the marker ink. Dirk 14:35, 25 February 2009 (EST)
• spin for 90 seconds, full speed to dry.
• elute with 50 ul water into a fresh Eppendorf tube

EcoRI/BamHI Digestion of Construct 19 synthesis product

• Set up the following reaction:
• 8uL of eluted PCR product
• 1uL of NEB Buffer 2
• 0.5uL EcoRI
• 0.5uL BamHI
• Incubate at 37 degrees on the thermocycler for 1hr
• Incubation carried out for only 40 minutes Dirk 15:27, 25 February 2009 (EST)
• Add 250uL of isopropanol and mix
• This was incorrect - now, instead of gel purification, a small fragment clean up will be carried out. Dirk 15:38, 25 February 2009 (EST)
• Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
• This step was not carried out because of the incorrect addition of IPA to the mixture prior to running the gel. Small fragment clean up carried out instead. Dirk 15:38, 25 February 2009 (EST)

Emergency Small-Frag Zymo Cleanup of Construct 19

• Transfer DNA and IPA mix into the Zymo column (small clear guys)
• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Solution went dark yellow upon addition of ADB buffer. Dirk 15:45, 25 February 2009 (EST)
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• spin for 90 seconds, full speed to dry.
• elute with 50 ul water into a fresh Eppendorf tube

## Dirk 13:24, 23 February 2009 (EST)

```Summary of activities:
* There was no time to carry out the digestion and second clean up of Construct 18
* Construct 18 is eluted in water in an Eppendorf tube
* Construct 18 had a blue liquid at the bottom of the collection tube after the drying step in the first Zymo Cleanup
* Constructs 17 and 19 have been handed over to Gabriel in three tubes bound by red tape for PCR

To do today:
* Redo the PCR for Construct 17 {<ECOHLY>} (see entry for 18 Feb 2009)
* Cleanup, digest, and cleanup for Construct 18 {<AG4>}
* Run second PCR for Construct 19 {<CBK>}
```

### Construct 17 {<Beta Roll>}

Dilute oligonucleotides for construct 17

• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 17 A (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007F (10uM)
• 1 uL Oligo Odvs008rR (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uL Template DNA E.coli 0157:H7
• Run this PCR on program 55

Prepare the PCR reaction for construct 17 B (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007R (10uM)
• 1 uL Oligo Odvs008rF (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uLTemplate DNA E.coli 0157:H7
• Run this PCR on program 55

### Construct 18 {<AG4>}

Cleanup, digest, and then cleanup again of construct 18.

First Small-Frag Zymo Cleanup of Construct 18

• Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
• Transfer into the Zymo column (small clear guys)
• Add 500uL of Ethanol and pipette up and down to mix
• Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
• Add 200 uL of PE or Zymo Wash buffer
• spin for 90 seconds, full speed to dry.
• A small amount of blue liquid was present in the collection tube at the end of this step. Dirk 15:38, 23 February 2009 (EST)
• elute with 50 ul water into a fresh Eppendorf tube

### Construct 19 {<CBK>}

Dilute oligonucleotides for construct 19

• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the second PCR reaction for construct 19 (Synthesis)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs003 (10uM)
• 1 uL Oligo Odvs006 (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uL Template DNA
• Run this PCR on program 55

## Dirk 13:21, 18 February 2009 (EST)

Today, I set up the PCR reactions for the three parts.

Dilute oligonucleotides for construct 17

• Add to the tube a volume of ddH20 10x ul the molar amount of DNA
• Mix and spin
• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 17 A (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007F (10uM)
• 1 uL Oligo Odvs008rR (10uM)
• 0.5 uL Expand Polymerase 1
• Run this PCR on program 55
• FORGOT TO ADD TEMPLATE DNA Dirk 16:03, 18 February 2009 (EST)

Prepare the PCR reaction for construct 17 B (Restriction site removal)

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007R (10uM)
• 1 uL Oligo Odvs008rF (10uM)
• 0.5 uL Expand Polymerase 1
• Run this PCR on program 55
• FORGOT TO ADD TEMPLATE DNA Dirk 16:03, 18 February 2009 (EST)

Dilute oligonucleotides for construct 18

• Add to the tube a volume of ddH20 10x ul the molar amount of DNA
• Mix and spin

Prepare PCR reaction for construct 18 (Wobble)

• 29 uL water
• 5 uL Expand 10x Buffer 2
• 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 5 uL Oligo Odvs002F (100uM)
• 5 uL Oligo Odvs002R (100uM)
• 0.75 uL Expand Polymerase 1

Dilute oligonucleotides for construct 19

• Add to the tube a volume of ddH20 10x ul the molar amount of DNA
• Mix and spin
• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Prepare the PCR reaction for construct 19 (Synthesis)

• 22 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs003 (10uM)
• 1 uL Oligo Odvs004 (10uM)
• 1 uL Oligo Odvs005 (10uM)
• 1 uL Oligo Odvs006 (10uM)
• 0.5 uL Expand Polymerase 1
• Run this PCR on program 55
```Will have to redo construct 17 because I forgot to add the template DNA to the reactions.
```

## Dirk 00:39, 10 February 2009 (EST)

Carried out the oligos and construction file for the Cellulose Binding Knottin CBK (Bdvs003, or M10046).

## Dirk 00:17, 10 February 2009 (EST)

Carried out the oligos and construction file for the Silver-binding peptide AG4 (Bdvs002, or M10023).

```* Design Oligos and make construction file for Cellulse Binding Knottin (M10046)
```

## Dirk 15:01, 9 February 2009 (EST)

Got the project ball on Wednesday last week. Assigned project 15954 - Beta Roll and Silver Peptide. Also assigned Cellulose Binding Knottin, from 37738. The project page can be found here (SBB09_15954).

Spent the lab time designing the oligos and writing the construction file for the Beta Roll (Bdvs001, or M10022).

```* Design oligos and make construction file for Silver Peptide (M10023)
* Design Oligos and make construction file for Cellulse Binding Knottin (M10046)
```