Extraction of Plasmid DNA from bacteria (Alkaline lysis method)

Principle:

The bacterial cells are lysed with lysozyme and SDS at high pH and the lysate is then neutralized. The plasmid undergoes renaturation but not the chromosomal DNA. The later gets precipitated out in the form of protein-DNA-SDS complex. Subsequent deproteinization with phenol: chloroform, plasmid DNA is precipitated with ethanol by spinning at high speed. 

Requirements:

1. Overnight Log- phase culture in LB broth (10 g tryptone, 5 g yeast extract and 10 g NaCl, 1L DW)
2. Solution I (Lysis buffer): 25 mM Tris, 50 mM Glucose, Lysozyme (GNB: 0.5 mg/ml, GPB: 3-5 mg/ml), pH 8.0
3. Solution II (Lysis): 0.2M NaOH, 1% SDS 
4. Solution III: 3 M Sodium acetate, pH 4.8 by acetic acid
5. Phenol: Chloroform (1:1)
6. RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0)
7. Absolute ethanol/Isopropanol
8. 70% ethanol
9. TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0; Autoclave before use
10. Water bath
11. Cooling centrifuge (upto 20000 rpm)
12. Micropipettes (1 – 10, 10 -100, 100 – 1000 µl ) and sterile tips

 Procedure: 

1. Take 1.5 ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000 rpm for 2 min.
2. Remove the supernatant and spin once with same volume as above to collect more cell mass.
3. Remove the supernatant.
4. Add 100 µl Sol. I. Re-suspend and keep for 30 min at RT.
5. Add 200 µl freshly prepared Sol. II and mix gently (Do not vortex). Keep for 5 min at 4°C.
6. Add 150 µl ice cold Sol. III and mix gently. Keep in ice bath for 10 min.
7. Spin at 10000 rpm for 5 min at 4°C. Collect Sup-T into new MFT.
8. Add 1-2 µl of RNase and incubate for 10 min at RT.
9. Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10 min at RT.
10. Centrifuge at 8000 rpm for 10 min at 4ºC and collect the supernatant in a new sterile MFT.
11. Add two vol. of absolute ethanol and stand it for 10 min in cold.
12. Spin at 13000 rpm at 4ºC for 10 min.
13. Remove the Sup-T into new MFT and add 1000 µl of 70% ethanol. Mix well and keep at 4°C for 30 min.
14. Spin at 13000 rpm at 4ºC for 10 min. Remove Sup-T.
15. Dissolve the pellet collected during spin in 50 µl TE and store in deep freeze.

Solid-State Drive (SSD)

An SSD (solid-state drive) is a type of nonvolatile storage media that stores persistent data on solid-state flash memory and has no moving parts unlike a hard disk drive (HDD), which stores data on a spinning disk. Two key components make up an SSD: a flash controller and NAND flash memory chips. The architectural configuration of the SSD controller is optimized to deliver high read and write performance for both sequential and random data requests. SSDs are sometimes referred to as flash drives or solid-state disks.

 To prevent volatility, SSD manufacturers design the devices with floating gate transistors (FGRs) to hold the electrical charge. This allows an SSD to retain stored data even when it is not connected to a power source. Each FGR contains a single bit of data, designated either as a 1 for a charged cell or a 0 if the cell has no electrical charge.

Unlike a hard disk drive (HDD), an SSD has no moving parts to break or spin up or down. A traditional HDD consists of a spinning disk with a read/write head on a mechanical arm called an actuator. The HDD mechanism and hard disk are packaged as an integrated unit. Businesses and computer manufacturers have used spinning disk historically, owing to their lower unit cost and higher average durability, although SSDs are now common in desktop and laptop PCs.

Things Should Know before you Buy SSD

1. SSD Disk Capacity

When you buy SSD for your laptop or Desktop, you can keep your SSD for OS and Apps and secondary HDD for your data in a Dual Drive Configuration. A 40GB SSD will be enough to run your PC on Windows or MAC operating system with a couple of essential apps. But if you can afford 80GB SSD, that will be a decent size and no need to worry about low memory for a while.

You can’t go with a smaller size if you are planning to replace your entire HDD with new SSD (Single Drive Configuration). An SSD with 250GB should be the starting point for your computer to take care of your OS, Application, and Data. There are SSDs that comes in Terabytes if you are ready to burn some additional bucks.

2. SSD Performance

Manufacturers are specifying the SSD performance related to the Sequential Read, and Sequential Write speed that typically goes up to 500MB/s in reading and bit lower in write.

3. SSD Data Random Transfer Rate:

SSD Random Write Speed and Random Read Speed is another benchmark to measure the performance of SSD. Random Read/Write Testing performs with small blocks of at random locations on the drive. Naturally, this process will be slow compared to Sequential Data handling. Typically, around 25% read-write operation will be random for an actual user.

4. SSD Flash Memory:

Solid state drives are based on flash memories with different level of NAND memories. The table below will give us a quick review of technologies those are available in the market now.

5. SSD Endurance MTBF (Reliability):

Mean Time Between Failures (MTBF) is the manufacturer’s estimate of total running hours of product shipped divided by the number of failed units. Long MTBF is always useful indication but no guarantee that the product can last that long. The reliability for SSDs falls in the range of a couple of million hours.

6. SSD Hardware Interface:

Most of the SSDs are coming with built-in Serial ATA (SATA) interface SATA support. The transfer speed can vary on the SATA versions. The new SSDs support up to SATA III that offers 6GB transfer speed where SATA II is capable of transferring 3GB where SATA I is limited to 1.5GB in data transfer.