2012-04-06T10:53:00.001-07:00

Closmid Cloning

Cat no.WEBC931 & TNC9401

A insert DNA Prperation
B Insert DNA End -Repair Reaction

For DNA

4 ul 10 X end repair buffer
4 ul 205 mm dntp mix
4 ul 1mm Atp
no D/W

24 ug sheared insert DNA

4 ul end repair enzyme mix

finally 40 ul

for Control

4 ul 10 X end repair buffer
4 ul 205 mm dntp mix
4 ul 1mm Atp

19 ul D/W

5 ul Cosmid insert

4 ul end repair enzyme mix

finally 40 ul

Add 6x loading buffer
incubate 70`c 10 min
put in freeze for 10 min
make gel without adding ETBR
load 8ul of sample ,mark it
like
C D C C C C D x
1 2 3 4 5 6 7 8
cut 1, 2& 7 where in 8 there is no sample
take out 3,4,5,6 then check in 1,2 & 7 where the bane is present
mark the side where the DNA is present in C from 1or C which is present in 1st
cut approximatelly 4 mm pices of agar rose
make sure that tse pices is not on the band it some far away from upper & lower part
cut small pices and put in freeze

Himedia Protocol + Modified & TAE & TBE Buffer

2012-04-03T14:56:00.002-07:00

Hi Media

MB-542

For DNA Isolation Purification For Difficult Siol Samples Follew Alrternate

Protocol

Lysis

To the Hibead Tube (DBCAO4) Provide add 750ul of soil Lysis solution(SL)(DS0079) & 250-500mg of Soil Sample mix by inverting the tubes Several times or by gentelly vortex
Secure the hibead tube horizontelly an a flat -bed vortex pad using a tape & vortex at maximum speed for 10 minutes.
Ensure that hibead tubes rotate freely i the centrifuge without rubbing centrifugre the tube at 13,000 x g (~ 14,000rpm for 1 minute at room temperature.

(NOTE = makesure nott be exceed the speed more than 13,000 x g or else the tubes may breath)
Transfer the supernant new 2.0 ml collection tube (not provided)

(NOTE = the supernant may still contain some soil particles)
Add 250ul of inhibitor Removal Solution (IRSH)(DS006) Vortex for 5 sec & incubate at 4`C for 5 minutes.
Centrifuge the tube for 1 minutes at 10,000 x g (= 12,000 rpm) at room temperatre

Binding
Transfer the superntant to a clean 2.0 ml collection add 1.2 ml Binding Solution(SB) (D50067) & vortex for 5 sec
Load onto hielute Miniprep Spin Column(DBCAG) load approximately 650 ul of the Lysate on the elute miniprep Spin Column & Centrifuge for 1 min at 10,000 x g ( 12,000 rpm) at room temp Discard the flow through load another 650 ul on the column & repeat the centrifuge .Discard the flow-through load the remaning lysate on the column & Cebtrifuge as before discard the foiw-through.

WASH
( Prepare wash soln as indicate in general preperation instruction) Add 500 ul of dillutr wash solution & centrifuge at 10,000 x g ( = 12, 000 rpm) at room temperature for 1 minute ,Discard the flow through repeat the wash flow through repeat the wash step more times.
Discard yhe flow centrifuge flow through and centrifuge the column at 12,000 x g ( = 13,000 rpm) at room temp for an additional 1 minute to remove any residual ethanol

DNA Elution
Transfer the column to new 2.0 ml Collection tube & add 100 ul of Elution buffer (ET) (D50080) or nolecular biology Gradew Water (ML024) directly on to center of the column member centrifuge the tube for 1 mmin at 10,000 x g ( - 12,000 rpm) at room temp.

Modified Protocol

MB -542

Take Different salt concentration like 5%,2.5%, 1.25%,0.625% & Finally 0%

from the protocol the result is present in 2.5%,1.25% & 0 %

but godd result is seeen in 2.5%

So as per the protocol take 750 ul 0f soil lysis solution in hibead tube with 2.5 % salt concentration
Add 500 mg 0g soil mix well & vortex for 10 min
Incudate for 95`c for 10 min
centrifuge the tube for 10- 15 min at room trmp
collect superntant in another tube and put in freeze at 4`c for 5 min

[NOTE: add IRSH solution depeno on superntant amount ]
add 250 ul if IRSH solution & Centrifuge for 10 min at room temperature
Add 1.2 ml of Bindibg Solution(BS) with IRSH Solution
vortex for 10 minutes
transfer 650 ul of solution in mini prep filter
Centrifuge tube for 5 min full speed
discard the lower liquid from the tube & repeat the Same step for 2 times[ finally three times]
Add 500ul of wash solution in mini prep filter repeat the step for 2 times
discard the lower liquid and centrifuge tube for 5 minutes

[ without any solution]
transfer the miniprep filter in new 2.0 ml collection
take 100ul of Elution buffer and load at the center if the filter and centrifuge for 10 minute at room temp
load 2ul sample +2ul of loading dye mix & load in gel

preperation of TBE Buffer

Component		10x Stock Amuont	1x Stock Amount
Tris Base	89 mm	108.0 gm	10.8 gm
Boric Acid	89 mm	55.00 gm	5.5 gm
EDTA(pH-8)	2 mm	7.44 gm	0.74 gm

Dissolve Appropriate amount in required amount
For preperaing 1x = take 1X Solution And Dissolve in 1000 ml DDW (Autoclaved)

Simple Preperation of TAE Bffer
STEP	Solution -1	Solution -2
0	Final Preperation 186.21mg/ml=0.5 M	242g =tris Buffer [2M] 57.1ml = Acetic Acid [2M] __________________________ For 250 ml Volume
1	18.62gm= Na2EDTA 80 ml = Sterile DD/W Set pH = 8 with NaoH Set Final Volume 100 ml	g tris 150 ml DD/W 14.27 Acetic acid ___________
2		Add 25 ml Solution -1
3 Make total Volume 250 ml with adding DD/W
For Preperation of 1X take 20 ml Stock Solution And 4800 ml DD/W Sterile Mix Well and Used as Buffer
Finally Buffer is in the 1X

DD

Himedia Protocol + Modified & TAE & TBE Buffer