Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics"

1 PCR Amplification of MaSp Plasmids

∙ Set up four 25 uL PCR reactions to test amplification. Used two pairs of primers and two different cycling conditions.

∙ Used MaSp2 2CA as template. Diluted 1:1000 to get 219 pg/uL. Created 2 master-mixes, one with each primer pair.

∙ Tested VF2/VR primers and post-elution primers using 17 cycles and 15 cycles using protocol below:

∙ We used 66 C as annealing because that temperature worked for previous amplification with the post-elution primers. In addition, NEB Tm calculator indicates 66 C annealing for the VF2/VR primer pair.

2 Results

∙ Cast 2% TAE gel to visualize results. Used 2 uL of NEB 50 bp ladder.

∙ The VF/R primer pair amplifies our plasmid as expected, but the post-elution primers do not. The expected size for VF/R amplification is 443 bp.

∙ May be due to bad primers for post-elution. Re-order or remake? We need to find more of the post-elution primers.

1 NotI Concatemers

Used yesterday’s gel purified fragments. We performed ligation using a 5:1 ratio of insert to vector. Our vector size is 2046 bp. Our insert is 143 bp. For 1121 ng of vector, we need 391.75 ng of insert. We set up the ligation reaction as below:

We used excess insert because the recorded concentration from the nanodrop was higher than the theoretical yield. In addition, we used more T4 Ligase than in typical reactions due to the reaction having three times as many sticky ends to ligate.

2 Results

We cast a 0.8% TAE agarose gel to separate the bands. We ran this particular gel at 90 V for 2 hours. We excised four bands indicated for gel purification (Qiagen). The bottom most band may be due to vector self-ligation. The second band at 2.2 kb is probably singly ligated product.

In the future, we should CIP treat the vector so it doesn’t re-ligate.

1 Gel Extraction

Performed gel extraction (Qiagen kit) of bands excised from yesterday’s gel. Results below:

2 2AB, 2BC, 2CA BsaI Ligation Test

Set up ligation tests for remaining conditions (see entry for 5/8/2015)for other tests. For each reaction, we used 50 ng of each DNA species.

2.1 T4 Ligase Reactions

Reactions were incubated at 25 C for 10 min.

2.2 T7 Ligase Reactions

Reactions were incubated at 25 C for 10 min.

3 Results

We cast a 2% TAE agarose gel to visualize the results. We used 2 uL of 50 bp ladder. See below.

1 2AB, 2BC, 2CA Ligation Test

1.1 BsaI digestion of 2AB, 2BC, 2CA

Used 5 ug of each DNA.

We are conducting ligation tests between all possible combinations using T4 and T7 ligase

2 NotI Concatemers

Sri suggested using NotI to digest the plasmids and create a super plasmid that contains multiple copies of the monomers.
Testing NotI digestion today using 2CA with the protocol below.

3 Results

Cast 200 mL of a 1.5% TAE agarose gel to visualize results of both BsaI and Not1 digestion.

1 T4/T7 Ligation Test

We tested the ability for the BsaI digested and gel purified monomers to ligate together. We ran these initial tests using both T4 and T7 DNA ligase using the following protocols (50 ng for each DNA species):

1.1 T4 Ligation

1.2 T7 Ligation

2 Results

We cast a 2% TAE gel to visualize the results. We use 2 uL of NEB 50 bp ladder. The gel was run at 150V for 35 min.

Our ligation test indicates that the new sticky ends exhibit specific binding. In both reactions, the ligation was incomplete due to the presence of a band at 100 bp. This may be due to non-equal amounts of DNA.

We plan to conduct this same test next week using all possible combinations between the three monomers to verify specific binding. We plan to test both T4 and T7.

1 BsaI Digestion of 2AB, 2BC, 2CA

∙ Gel purification of yesterday’s BsaI digested fragments (Qiagen).

∙ Obtained approx. 7 ng/uL for each monomer. A 260/280 for all three were around 1.8. Will use these as is for ligation test tomorrow.

∙ Spun down yesterday’s cells in 50 mL tubes for 15 min @ 6000x g @ 4 C.

∙ Used Maxiprep (Qiagen) kit to prepare DNA. Eluted in 400 uL EB. Concentrations from each are listed below:

∙ The DNA amounts are low. We probably need to improve our technique.

∙ Miniprep (Zymo) yesterday’s overnight bacterial culture. We also created glycerol stocks from these cultures.

∙ Prepared 100 mL culture of 2AB, 2BC, 2CA for Maxiprep tomorrow.

1 BsaI Digestion Test for 2AB

Tested digestion of 5 ug of plasmid to see efficiency of digestion.

Results are shown below:

∙ The digestion process works, so we’ll be proceeding with this protocol.

∙ We gel extracted the digested insert band. The end result was 11.9 ng/uL, A (260/280): 4.4

2 BsaI Digestion for Post-elution primer amplified MaSp

We BsaI digested the PCR products from previous PCRs using the following protocol

2.1 BsaI Digest Protocol

*For 2BC, we combined 8.84 uL of 33.9 ng/uL solution and 9.17 uL and 33.9 ng/uL solution to get 500 ng total.

2.2 Purification

∙ PCR purified the post-elution PCR digestion products and got the following yields:

∙ Yield and absorbance for the digested inserts are not very good. This might be a problem due to the PCR process, even though product was generated.

3 Bacterial Propagation

∙ Grew 7 mL each of AB, BC, CA from glycerol stock for DNA extraction tomorrow.

1 Calculation for Primer exhaustion

∙ Vector+insert = 2205 bp (150 bp insert) (2055 bp vector)

∙ DNA: 649 g/mole/bp

∙ AB: 143105 g/mole

∙ 500 pg / 1431045 g/mol = 3.494 E -16 moles DNA

∙ 1.25 uL of 10 uM primers = 1.25 E -11 moles primers

∙ 1.25 E -11 / 3.494 E -16 = 35775.6

∙ log2(35775.6) = 15.1 cycles (assuming perfect PCR)

2 Modified PCR using post-elution primers

Redo PCR using same amounts of reagents, but with 20 cycles instead of 30. Made 2x 50 uL for each MaSp plasmid.

1 Post-elution primer PCR of MaSp plasmids

∙ The previous anomalous results may be due to a human factor. So we decided to have Olivia prepare the PCR today using the same protocol, but scaled to 3x 25 uL each for 2AB and 2BC. Results shown below:

1 Post-Elution primer PCR of MaSp 2 plasmids

∙ Performed same PCR reaction as yesterday, but with 2x 50 uL for each reaction. This was prepared as a master mix. Results shown below.

∙ We excised the bands from 2AB, 2BC (only one), and 2CA.

1 Post-Elution Primer PCR Amplification of MaSp2 Sequences

We used the post-elution primers to test amplify 2AB plasmid and to determine an appropriate annealing temperature. We used the following protocol:

2 Results

∙ Base on these results, we decided to scale up to 3x 50 uL reactions for each monomer, and would use 66 C as the annealing temperature.

3 Amplification Again

We scaled up the above protocol for 3x 50 uL reactions for each monomer.

4 Results for second PCR

∙ Visualized on 2% gel. Used 2 uL 50 bp ladder.

1 2AB, 2BC, 2CA Sticky End Ligation Test

∙ The ligation test was run on 2% gel with 2 uL of 50 bp ladder. Results shown below.

1 Sequencing of MaSp2 constructs

∙ Prepared each sample from yesterday for sequencing.

∙ Diluted 500 ng of each DNA species and 2.5 uL of VF2 primer into 15 uL in a PCR strip.

2 Results: 4/25/2015

∙ Used Clustal W2 to align sequencing results.

∙ Constructs 2AB-2, 2BC-1, and 2CA-2 are sequenced properly.

1 Miniprep

∙ Prepared Glycerol stocks of each culture: 500 uL of bacteria + 500 uL 50% v/v glycerol.

∙ Used zymo miniprep kit to extract DNA from yesterday’s cells.

1 Bacterial Transformation

∙ Observed many colonies growing on the plates. We will do colony PCR to verify proper insertion.

2 Colony PCR

∙ Picked four colonies from each plate. Resuspended each each colony in 100 uL water. Used protocol below:

3 Results

∙ Cast 1.0% gel to visualize.

∙ We picked colonies 3 and 4 from each construct and grew 5 mL liquid culture for miniprep and sequencing tomorrow. Renumbered the cultures to 1 and 2 respectively.

4 Column purification of MaSp2 PCA

∙ Used zymo kit to purify yesterday’s MaSp2 PCA reactions. Eluted in 10 uL EB. Results below:

5 BsaI Digestion of PCA products from 4/21/2015

∙ Set up 2x 25 uL reactions for each:

∙ The digestion products were column purified using zymo kit with the following yields:

1 Ligation of MaSp 2AB, 2BC, 2CA in to pSB1C3

∙ Used ligation calculator to determine amounts of each insert to use to ligate into 50 ng of vector for a 3:1 ratio of insert to vector.

∙ 2000 bp vector, 170 bp insert.

∙ Determined 12.75 ng of insert to 50 ng vector. Proceeded with ligation using protocol below:

2 Bacterial Transformation

∙ Used 2 uL of each reaction to transform 50 uL competent cells.

∙ Incubate on ice 5 min.

∙ Add 200 uL of pre-warmed SOC to each tube.

∙ Rescue for 10 min.

∙ Plate 50 uL onto chloramphenicol plates. Incubate at 37 C overnight.

3 MaSp2 PCA

∙ Set up 2x 50 uL reactions for AB, BC, CA each using protocol from 4/13/2015 scaled to 50 uL with the following modifications:

∙ Annealing temp: 64 C

∙ 25 cycles instead of 20.

1 PCR Amplification of pSB1C3

∙ Due to poor amplification of pSB1C3 from friday, we are doing a temperature test today.

∙ Followed same protocol as 4/17/2015, but scaled to 3x 25 uL reactions.

∙ Decided to take nanodrop reading of the template Julian provided. It was 230 ng/uL. Instead of using stock concentration of template, we will use a 1:100 dilution. Results shown below.

∙ We gel extracted the bands together. 26.4 ng/uL, A 260/280: 1.56.

2 EcoRI and PstI Digestion of MaSp 2AB, 2BC, 2CA, and pSB1C3

∙ We pooled together all the linearized pSB1C3 that we had. Final concentration: 16.5 ng/uL.

∙ Set up digestion as below:

∙ Column purified all DNA after digestion using Zymo kit. Eluted in 10 uL of EB. Results shown below:

1 PCR amplification of pSB1C3

We set up a 50 uL reaction using the following protocol from Julian’s notebook.

2 Results

∙ Cast a 1% gel to visualize.

∙ Gel extraction (Qiagen) yielded 36.47 ng/uL, A 260/280: 1.91.

1 Polymerase Chain Assembly of MaSp 2AB sequence.

Repeated PCA of 2AB using protocol from 4/13/2015 with the follow modifications.

∙ Annealing Temp: 64 C

∙ 25 cycles instead of 20.

∙ We cast a 1.5% gel to visualize the results shown below.

2 Gel Extraction

Fasih gel extracted the two 2AB bands.

1 MaSp2 Polymerase Chain Assembly

Set up two 50 uL PCR reactions each for 2BC and 2CA using the same guidelines on 4/13/2015 with the following modifications:

∙ Annealing Temp: 64 C

∙ 25 cycles instead of 20

2 Gel Purification of MaSp2 Monomers

∙ Visualized with 1.5% agarose gel. Similar result to yesterday’s gel.

∙ Used gel purification kit (zymo) to extract DNA from 2AB, 2BC, and 2CA.

1 MaSp2 Polymerase Chain Assembly

Set up 2 50 uL reactions following the same protocol from yesterday scaled to 50 uL for 2AB. We included the following modifications:

∙ Annealing temperature: 64 C

∙ 25 cycles instead of 20

∙ Used primers 2A-F, 2B-R

2 Results

Cast 1.5% TAE agarose gel. Used 3 uL of NEB 50 bp ladder.

This protocol works better than the one previously. The target product at 170 bp is clearly more intense. While there is still some primer dimer formation, we will stick with this protocol.

1 Polymerase Chain Assembly

The primers corresponding to the new MaSp2 sequences with a valine added to the end came today. We conducted polymerase chain assembly to place the biobrick prefix/suffix and the BsaI restriction enzyme recognition sites with the designed sticky ends.

We used NEB’s Q5 PCR annealing temperature calculator to determine that an annealing temperature of 65 C was optimal. We also decided to test annealing at 61.4 C and 59 C. In addition, we planned to use the GC enhancer because our sequence is GC-rich. We prepared reactions as follows:

2 Results

We cast a 1.5% TAE gel to visualize the results. We used 2 uL of NEB 50 bp ladder.

Our results indicate that 65 degrees is too high, and does not have good yield, but 61.4 is too low and has non-specific amplification as shown by the smears. The band at 50 bp is attributed to primer dimer formation.

Based on our results, tomorrow, we will perform polymerase chain assembly with annealing at 64 C and with 25 cycles.