Various Lysing Methods with D.aquaticus 7kb Primers

 Introduction 

Recently, we had obtained E. coli's cm^r gene from pRAD1. However, when observing a gel for 7kb fragments, only primer bands were shown. Three hypotheses were that the master mix wasn't made correctly, the annealing temperature during PCR was too high for binding, and the primers could have been constructed incorrectly. To test these hypotheses, we'll run four long amplification PCR reactions. The two templates we'll use are boiled D.aquaticus cells and a plasmid extraction sample of D.aquaticus that was used when first constructing the current 7kb primers. Each pair will use m23 primers and 7kb primers. The purpose of the m23 primers is to be used as a control for master mix. The m23 primers are expected to prime to the m23 portion of D. aquaticus' 7kb plasmid and show on the agarose gel. We'll also lower our annealing temperature to 57°C and expect more non-specific binding than from our 59°C samples. 


Procedure

To begin making our 4 25ul long amplification PCR reactions, 20ul of long amplification mastermix is added to the 4 pcr tubes. Then, 1ul of forward and 1ul of reverse m23 primers are added to 2 tubes. We then added 1ul of forward and reverse 7kb primers to the other 2 PCR tubes. Next, 100ul of D. aquaticus culture is boiled for 15 minutes at 95°C in a thermal cycler. After setting the culture on ice for 1 minute, 3ul of the boiled culture is added to 1 tube containing 7kb primers and 1 tube containing m23 primers. 3ul of the plasmid extraction template is then added to the other reactions for a total of 4 25ul PCR reactions. Lastly, PCR reactions were read on the nanodrop spectrophotometer and ran on a 1% agarose gel with a 10:2 template to dye ratio. 


Results








Agarose gel containing bands at ~700 bp




Discussion

The m23 bands showing at around 700bp indicate that our master mix isn't a current issue. Additionally, boiled cells may be a preferred template, as the band is significantly brighter than the plasmid extraction band. Despite lowering the annealing temperature, no bands at 7kb were seen for either of the templates used. Two hypotheses are that our initial denaturing time isn't long enough or that the annealing temperature isn't in the correct range. To test these hypotheses, we will extend our initial denaturing time from 30 seconds to 4 minutes, as well as perform a heat gradient PCR reaction with boiled cells as our template. The range that we'll run our heat gradient by is 50-66°C. Bands will indicate whether or not our 7kb primers are constructed correctly.


Comments

Post a Comment

Popular posts from this blog

Colony PCR Amplification of 7kb Plasmid from Deinococcus aquaticus and cmr from E. coli's pRAD1 Plasmid

Image
 Introduction  This semester, Leilani Boren, Micheal Dockham and I are transforming the 7kb plasmid from Deinococcus aquaticus with E.coli's chloramphenicol resistance gene via PCR amplification and Gibson assembly. Once assembled, the 7kb plasmid will be farmed and reintroduced into D. aquaticus. To do this, we must first prime D.aquaticus' 7kb plasmid as our vector and E. coli's chloramphenicol resistance gene as our antibiotic resistance gene via PCR.  DNA cloning works by inserting a target gene (cmr) into a plasmid (7kb) . Afterward, the plasmid is introduced into bacteria (E.coli) via transformation. Colonies will be screened via selective media (LB + chloramphenicol) and, once verified, will be used to farm recombinant plasmid DNA.  Typically, Plasmid DNA is manipulated via restriction enzymes which will cut at cloning sites to create an area for our fragment to be introduced and hybridized. However, we will be using the Gibson Assembly due to successes in combining
Read more

Restriction Enzyme on Deinococcus Aquaticus 7kb Fragment

Image
 Introduction Last week in lab, two largely concentrated samples of the 7kb fragment from Deinococcus Aquaticus was obtained. However, the gel showed this sample to be at 14kb. A theory for this is that two 7kb fragments are mineralized together. Therefore, we are using restriction enzyme to demineralize these fragments and have them appear at 7kb. If this isn't the case, we will use primers. Restriction enzymes are sequences that are recognized by restriction enzyme to cut plasmids. We cut at the restriction site xbaI (1188 base pairs) but this should show as 7kb on the gel.  Procedure A 1mL tube was filled with 68ul pcr water, 10ul cutsmart buffer, 2ul restriction enzyme , and 20ul of our plasmid isolation samples from the week prior (2mg). Once we have a 100ul total , we will incubate this tube on a heat block at 37 degrees celcius. Due to not knowing how long this enzyme will take to completely cut at all of the restriction sites, we will use 4 incubation time periods. These ti
Read more

Using New GelRed on Edited 7kb PCR Reactions and Extractions

Image
 Introduction  Recently, a heat gradient from 50 °C-66 °C   on the 7kb plasmid from D.aquaticus was ran and contained no visible banding. Due to an empty agarose gel, this may reveal that the template we're using ( boiled cells ) doesn't contain the 7kb plasmid. This would mean our primers aren't able to bind appropriately. Another possibility is that our annealing temperature. Therefore, the alternative bead-beating template will be used in a PCR reaction and run on the more sensitive GelRed agarose gel.  Additionally, a new lysing protocol involving proteinase k and lysozyme will be used to try to extract 7kb plasmid. A theory to test is that we've obtained 7kb plasmid and hadn't seen bands due to the previous dye needing >500ng of template DNA. This new GelRed should only need 5-50ng of DNA to bind visibly.  Methods To run LongAmp PCR products, PCR settings were set to the following: initial denaturing was set to 4 minutes at 94 °C, denaturing for 30 seconds a
Read more